Antibody neutralizing human respiratory syncytial virus

ABSTRACT

The present invention relates to monoclonal antibodies which have high anti-RSV neutralizing titers. The invention further provides for isolated nucleic acids encoding the antibodies of the invention and host cells transformed therewith. The invention yet further provides for diagnostic, prophylactic and therapeutic methods employing the antibodies and nucleic acids of the invention, particularly as a passive immunotherapy agent in infants and the elderly.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. Ser. No.16/434,729 filed Jun. 7, 2019, which claims benefit of divisionalapplication U.S. Pat. No. 10,358,480, issued Jul. 23, 2019, which claimsbenefit of U.S. Pat. No. 9,963,500 issued May 8, 2018, and which claimsbenefit of U.S. Provisional Patent Application No. 62/367,359 filed Jul.27, 2016, and U.S. Provisional Patent Application No. 62/247,841 filedOct. 29, 2015, each of which are incorporated herein by reference intheir entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “24158-US-CNT-2-SEQTXT-08DEC2020.txt”, creation date of Dec.4, 2020, and a size of 25 KB. This sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to human monoclonal antibodies which havehigh anti-RSV neutralizing titers, as well as the use of theseantibodies as a passive immunotherapy agent in infants and the elderly.

BACKGROUND OF THE INVENTION

Paramyxoviruses are enveloped negative-strand RNA viruses that aresignificant human and animal pathogens. Human Respiratory SyncytialVirus (hRSV, RSV) belongs to the family Paramyxoviridae, subfamilyPneumovirinae. Two subtypes, type A and type B, have been identified andare a major cause of severe and sometimes even fatal respiratoryinfections in children less than 6 months of age. Adults with underlyingdiseases, such as COPD, asthma, cancer, immunocompromised status,including HIV or post transplantation, are also at risk of developingsevere RSV infection. 15% of annual hospitalizations in adults over 50years due to acute respiratory infection are caused by RSV. In theUnited States, RSV causes more than 100,000 hospitalizations annually,and it is estimated to cause about 160,000 deaths globally each year.Currently there is no vaccine for RSV, and a trial with aformalin-inactivated virus was associated with increased diseaseseverity in infants upon infection with RSV. Other family membersincluding Human Metapneumo Virus (hMPV) and Human Parainfluenza Virus(hPIV) are also responsible for acute respiratory illness similar tohRSV.

The hRSV genome is a single-stranded negative-sense RNA molecule ofapproximately 15 kb that encodes 11 proteins. Two of these proteins arethe main surface glycoproteins of the virion. These are (i) theattachment (G) protein, which mediates virus binding to cells, and (ii)the fusion (F) protein, which promotes both fusion of the viral and cellmembranes at the initial stages of the infectious cycle and fusion ofthe membrane of infected cells with those of adjacent cells to formcharacteristic syncytia. The attachment protein G binds cellular surfacereceptors and interacts with F. This interaction triggers aconformational change in F to induce membrane fusion, thereby releasingthe viral ribonucleoprotein complex into the host cell cytoplasm.

Monoclonal antibodies against the F protein or the G protein have beenshown to have neutralizing effect in vitro and prophylactic effects invivo. See, e.g., Beeler and Coelingh 1989, J. Virol. 63:2941-50;Garcia-Barreno et al., 1989, J. Virol. 63:925-32; Taylor et al., 1984,Immunology 52: 137-142; Walsh et al., 1984, Infection and Immunity43:756-758; and U.S. Pat. Nos. 5,842,307 and 6,818,216. Neutralizingepitopes on the F glycoprotein were originally mapped by identifyingamino acids that were altered in antibody escape variants and byassessing antibody binding to RSV F-derived peptides. These studiesdemonstrated neutralizing antibodies are often targeted to two distinctlinear epitopes. See Graham et al., 2015, Curr Opin Immunol 35:30-38 fora review of the antigenic sites for the pre-fusion and post-fusion Fforms. Antigenic site II (also called site A) includes residues 255 to275 and is the target of palivizumab (SYNAGIS®, AstraZeneca). Thisepitope was predicted to be conformationally dependent, and thestructure of a more potent derivative of palivizumab in complex withthis epitope revealed that the linear epitope adopts a helix-loop-helixconformation. Antigenic site IV (also called site C) includes residues422 to 438 and is the target of antibodies MAb19 and 101F. This epitopeis C-terminal to the cysteine-rich region and is part of domain II,which in homologous paramyxovirus F glycoproteins remains structurallyunchanged between pre- and post-fusion conformations. 5C4, AM22 and D25delineate an epitope designated as site 0 which is only present on thepre-fusion F protein and were 50 times as potent as palivizumab. SeeMcLellan et al., 2013, Science 340:1113-1117; International PatentApplication No. WO 2008/147196 and U.S. Pat. No. 8,568,726. Other hRSVantibodies are described in International Patent Application Nos.WO94/06448 and WO92/04381 and U.S. Pat. No. 8,221,759.

An RSV vaccine for active immunization, if available, could not beutilized for the treatment of newborn babies with immature immunesystems or patients who are immunosuppressed. In patients whereprophylactic passive immunotherapy is required, as a result of a morechronic form of disease, current therapy is mediated via periodicintravenous inoculation of human IgG prepared from pooled plasma. Thistype of therapy, due to the low titers of neutralizing anti-RSVantibodies, involves a large quantity of globulin (e.g., 0.75 gm per kg)and consequently requires administration intravenously, in a clinic orhospital, over a lengthy period (2 to 4 hours), on a monthly basisduring the high risk months (fall, winter and early spring).

SUMMARY OF THE INVENTION

The invention provides anti-RSV F-protein antibodies and antigen bindingfragments thereof comprising the structural and functional featuresspecified below.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof that binds to human RSV F-protein, comprising: a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 3. In certain embodiments, the heavy chain or heavy chain variableregion does not comprise the amino acid sequence of SEQ ID NO: 9. In oneembodiment, the antibody or antigen binding fragment thereof optionallyhas at least one of the following characteristics: (i) binds to humanRSV pre-fusion F protein with a Kd value of about 1×10⁻⁹ M to about1×10⁻¹² M as determined by surface plasmon resonance (e.g., BIACORE) ora similar technique (e.g. KinExa or OCTET); or (ii) binds to human RSVpost-fusion F protein with a Kd value of about 1×10⁻⁶ M to about 1×10⁻⁹M as determined by surface plasmon resonance (e.g., BIACORE) or asimilar technique (e.g. KinExa or OCTET). In certain embodiments, theheavy chain comprises, consists essentially of, or consists of, theamino acid sequence of SEQ ID NO: 23.

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that binds to human RSV F-protein, comprising:a light chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 6. In certain embodiments, the light chain or light chainvariable region does not comprise the amino acid sequence of SEQ ID NO:8. In certain embodiments, the light chain is not associated with aheavy chain comprising the amino acid sequence of SEQ ID NO: 9. Incertain embodiments, the light chain is associated with a heavy chaincomprising the amino acid sequence of SEQ ID NO: 7. In one embodiment,the antibody or antigen binding fragment thereof optionally has at leastone of the following characteristics: (i) binds to human RSV pre-fusionF protein with a Kd value of about 1×10⁻⁹ M to about 1×10⁻¹² M asdetermined by surface plasmon resonance (e.g., BIACORE) or a similartechnique (e.g. KinExa or OCTET); or (ii) binds to human RSV post-fusionF protein with a Kd value of about 1×10⁻⁹ M to about 1×10⁻¹¹ M asdetermined by surface plasmon resonance (e.g., BIACORE) or a similartechnique (e.g. KinExa or OCTET) In certain embodiments, the light chaincomprises, consists essentially of, or consists of, the amino acidsequence of SEQ ID NO: 25.

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that binds to human RSV F-protein comprising:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; and (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 3. In certain embodiments, the heavy chain or heavy chain variableregion does not comprise the amino acid sequence of SEQ ID NO: 9. In oneembodiment, the antibody or antigen binding fragment thereof optionallyhas at least one of the following characteristics: (i) binds to humanRSV pre-fusion F protein with a Kd value of about 1×10⁻⁹ M to about1×10⁻¹² M as determined by surface plasmon resonance (e.g., BIACORE) ora similar technique (e.g. KinExa or OCTET); or (ii) binds to human RSVpost-fusion F protein with a Kd value of about 1×10⁻⁹ M to about 1×10⁻¹¹M as determined by surface plasmon resonance (e.g., BIACORE) or asimilar technique (e.g. KinExa or OCTET). In certain embodiments, theheavy chain comprises, consists essentially of, or consists of, theamino acid sequence of SEQ ID NO: 23.

In one embodiment, the antibody or antigen binding fragment thereofcomprises: (i) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 4; (ii) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (iii) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In certain embodiments, the light chain or light chain variableregion does not comprise the amino acid sequence of SEQ ID NO: 8. Incertain embodiments, the light chain is not associated with a heavychain comprising the amino acid sequence of SEQ ID NO: 9. In certainembodiments, the light chain is associated with a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 7. In one embodiment, the antibodyor antibody fragement thereof optionally has at least one of thefollowing characteristics: (i) binds to human RSV pre-fusion F proteinwith a Kd value of about 1×10⁻⁹ M to about 1×10⁻¹² M as determined bysurface plasmon resonance (e.g., BIACORE) or a similar technique (e.g.KinExa or OCTET); or (ii) binds to human RSV post-fusion F protein witha Kd value of about 1×10⁻⁹ M to about 1×10⁻¹¹ M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa orOCTET). In certain embodiments, the light chain comprises, consistsessentially of, or consists of, the amino acid sequence of SEQ ID NO:25.

In one embodiment, the antibody or antigen binding fragment thereofcomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3;(iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In certain embodiments, the heavy chain or heavy chain variableregion does not comprise the amino acid sequence of SEQ ID NO: 9. In oneembodiment, the antibody or antigen binding fragment thereof optionallyhas at least one of the following characteristics: (i) binds to humanRSV pre-fusion F protein with a Kd value of about 1×10⁻⁹ M to about1×10⁻¹² M as determined by surface plasmon resonance (e.g., BIACORE) ora similar technique (e.g. KinExa or OCTET); or (ii) binds to human RSVpost-fusion F protein with a Kd value of about 1×10⁻⁹ M to about 1×10⁻¹¹M as determined by surface plasmon resonance (e.g., BIACORE) or asimilar technique (e.g. KinExa or OCTET). In certain embodiments, theheavy chain comprises, consists essentially of, or consists of, theamino acid sequence of SEQ ID NO: 23 and the light chain comprises,consists essentially of, or consists of, the amino acid sequence of SEQID NO: 25.

In another embodiment, the invention provides an antibody or antigenbinding fragment that binds to human RSV F-protein comprising: (i) aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 4; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6; wherein the antibodyor antigen binding fragment thereof comprises a heavy chain variableregion comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity to aheavy chain variable region consisting of SEQ ID NO: 7 and a light chainvariable region comprising at least 90%, 95%, 96%, 97%, 98% or 99%identity to a light chain variable region consisting of SEQ ID NO: 8. Incertain embodiments, the heavy chain or heavy chain variable region doesnot comprise the amino acid sequence of SEQ ID NO: 9. In theseaforementioned embodiments, the sequence variations occur in theframework regions. In one embodiment, the antibody or antigen bindingfragment thereof optionally has at least one of the followingcharacteristics: (i) binds to human RSV pre-fusion F protein with a Kdvalue of about 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa orOCTET); or (ii) binds to human RSV post-fusion F protein with a Kd valueof about 1×10⁻⁹ M to about 1×10⁻¹¹ M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).In certain embodiments, the heavy chain comprises, consists essentiallyof, or consists of, the amino acid sequence of SEQ ID NO: 23 and thelight chain comprises, consists essentially of, or consists of, theamino acid sequence of SEQ ID NO: 25.

In another embodiment, the invention also provides an antibody orantigen binding fragment thereof that binds to human RSV comprising: (i)a heavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 4; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6. In certainembodiments, the heavy chain or heavy chain variable region does notcomprise the amino acid sequence of SEQ ID NO: 9. In one embodiment, theantibody or antigen binding fragment thereof comprises 1, 2 or 3 aminoacid substitutions in the heavy chain CDRs (SEQ ID NOs: 1-3) and/or inthe light chain CDRs (SEQ ID NOs: 4-6). The VH sequence of SEQ ID NO: 7has the CDRs of SEQ ID NOs:1-3; and the VL sequence of SEQ ID NO: 8 hasthe CDRs of SEQ ID NOs: 4-6. In one embodiment, the antibody or antigenbinding fragment thereof optionally has at least one of the followingcharacteristics: (i) binds to human RSV pre-fusion F protein with a Kdvalue of about 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa orOCTET); or (ii) binds to human RSV post-fusion F protein with a Kd valueof about 1×10⁻⁹ M to about 1×10⁻¹¹ M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).In certain embodiments, the heavy chain comprises, consists essentiallyof, or consists of, the amino acid sequence of SEQ ID NO: 23 and thelight chain comprises, consists essentially of, or consists of, theamino acid sequence of SEQ ID NO: 25.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof, comprising: a variable heavy chain comprising theamino acid sequence of SEQ ID NO: 7 and/or a variable light chaincomprising the amino acid sequence of SEQ ID NO: 8, wherein the antibodyor antigen binding fragment thereof binds to human RSV F protein. Inanother embodiment, the antibody or antigen binding fragment thereofcomprises a heavy chain comprising, consisting essentially of, orconsisting of, the amino acid sequence of SEQ ID NO: 23 and a lightchain comprising, consisting essentially of, or consisting of, the aminoacid sequence of SEQ ID NO: 25, wherein the antibody or antigen bindingfragment thereof binds to human RSV F protein. In one embodiment, theantibody or antigen binding fragment thereof optionally has at least oneof the following characteristics: (i) binds to human RSV pre-fusion Fprotein with a Kd value of about 1×10⁻⁹ M to about 1×10⁻¹² M asdetermined by surface plasmon resonance (e.g., BIACORE) or a similartechnique (e.g. KinExa or OCTET); or (ii) binds to human RSV post-fusionF protein with a Kd value of about 1×10⁻⁹ M to about 1×10⁻¹¹ M asdetermined by surface plasmon resonance (e.g., BIACORE) or a similartechnique (e.g. KinExa or OCTET).

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that binds to the same epitope of human RSV Fprotein as an antibody comprising the heavy chain of SEQ ID NO: 23 andthe light chain of SEQ ID NO: 25, wherein the antibody or antigenbinding fragment thereof has at least one of the followingcharacteristics: (i) binds to human RSV pre-fusion F protein with a Kdvalue of about 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa orOCTET); or (ii) binds to human RSV post-fusion F protein with a Kd valueof about 1×10⁻⁹ M to about 1×10⁻¹¹ M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).In one embodiment, the antibody comprises at least 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% sequence identity with the heavy chain variableregion and/or the light chain variable region of SEQ ID NOs: 7 and 8,respectively. In another embodiment, the antibody or antigen bindingfragment thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30amino acid substitutions in the heavy chain variable region of SEQ IDNO: 7 and/or the light chain variable region of SEQ ID NO: 8. In certainembodiments, the heavy chain or heavy chain variable region does notcomprise the amino acid sequence of SEQ ID NO: 9.

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that cross-blocks the binding of (or competeswith) an antibody comprising the heavy chain of SEQ ID NO: 23 and thelight chain of SEQ ID NO: 25 to human RSV, wherein the antibody orantigen binding fragment thereof has at least one of the followingcharacteristics: (i) binds to human RSV pre-fusion F protein with a Kdvalue of about 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa orOCTET); or (ii) binds to human RSV post-fusion F protein with a Kd valueof about 1×10⁻⁹ M to about 1×10⁻¹¹ M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).In one embodiment, the antibody or antigen binding fragment thereofcomprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity with the heavy chain variable region of SEQ ID NO: 7 or thelight chain variable region of SEQ ID NO: 8. In another embodiment, theantibody or antigen binding fragment thereof comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 amino acid substitutions in the heavy chainvariable region of SEQ ID NO: 7 or the light chain variable region ofSEQ ID NO: 8. In another embodiment, the antibody or antigen bindingfragment thereof comprises 1, 2 or 3 amino acid substitutions in theheavy chain CDRs (SEQ ID NOs: 1-3) and/or in the light chain CDRs (SEQID NOs: 4-6). In certain embodiments, the heavy chain or heavy chainvariable region does not comprise the amino acid sequence of SEQ ID NO:9.

In one embodiment, the invention relates to an isolated antibody orantigen binding fragment that binds to human RSV F protein comprising: aheavy chain comprising the amino acid sequence of SEQ ID NO: 7 orvariant thereof comprising up to 30 amino acid substitutions, and/or alight chain comprising the amino acid sequence of SEQ ID NO: 8comprising up to 12 amino acid substitutions. In certain embodiments,the heavy chain or heavy chain variable region does not comprise theamino acid sequence of SEQ ID NO: 9.

In certain embodiments, the invention relates to an isolated antibody orantigen binding fragment that binds to human RSV F protein, wherein theantibody binds to human RSV F protein through one or more of thefollowing interactions or all of the following interactions:

-   -   1) the light chain CDR3 loop, through residues Phe 91 and Leu        92, interacts with the side chain of Arg 429 of human RSV F        protein through the formation of two hydrogen bonds between the        carbonyl oxygens of Phe 91 and Leu 92 in the CDR3 loop and the        guanidino nitrogens of Arg 429 of human RSV F protein;    -   2) the light chain CDR2 loop, through residues Asp 50 and Glu        55, forms hydrogen bonds with Asn 426 and Lys 445 of human RSV F        protein;    -   3) the heavy chain CDR3 loop, through residues Tyr 104 and Tyr        110, form a surface for van der Waals interaction with Ile 432        on human RSV F protein;    -   4) the heavy chain CDR3 loop, through Asn 107, forms a hydrogen        bond with Lys 433 of human RSV F protein; and    -   5) the light chain packs against Glu 161 and Ser 182 of the        neighboring monomer of a RSV pre-fusion trimer.

In certain aspects of any of the above embodiments, the antibody orantigen binding fragment thereof is isolated.

In certain aspects of any of the above embodiments, the antibody orantigen binding fragment thereof is a recombinant antibody.

In certain aspects of any of the above embodiments, the antibody orantigen binding fragment thereof is a full-length antibody.

In certain aspects of any of the above mentioned embodiments, theantibody or antigen binding fragment thereof of the invention cancomprise a heavy region variable region consisting of: (a) any of thevariable heavy chains described above and (b) a leader peptide (forexample, the leader peptide of SEQ ID NO: 10). In certain aspects of anyof the above mentioned embodiments, the antibody or antigen bindingfragment thereof of the invention can comprise a light chain variableregion consisting of: (a) any of the variable light chains describedabove and (b) a leader peptide (for example, the leader peptide of SEQID NO: 10).

In certain aspects of any of the above mentioned embodiments, theantibody or antigen binding fragment thereof of the invention is anantibody comprising any of the variable heavy chains described above andany human heavy chain constant domain. In one embodiment, the antibodyor antigen binding fragment thereof of the invention is of the IgGisotype, and comprises a human IgG1, IgG2, IgG3 or IgG4 human heavychain constant domain. In one embodiment, the antibody or antigenbinding fragment thereof of the invention comprises a human heavy chainIgG1 constant domain wherein the IgG1 constant domain is afucosylated.

In certain aspects of any of the above mentioned embodiments, theantibody or antigen binding fragment thereof of the invention cancomprise any of the variable light chains described above and a humanlight chain constant domain. In one embodiment, the antibody or antigenbinding fragment thereof of the invention comprises a human kappa lightchain constant domain or a variant thereof, wherein the variantcomprises up to 20, 10, 5, 3, 2, or 1 modified amino acid substitutions.In another embodiment, the antibody or antigen binding fragment thereofof the invention comprises a human lambda light chain constant domain ora variant thereof, wherein the variant comprises up to 20, 10, 5, 3, 2,or 1 modified amino acid substitutions. In one embodiment, the antibodyor antigen binding fragment thereof of the invention comprises a humankappa light chain constant domain comprising the amino acid sequence ofSEQ ID NO: 14.

In one embodiment, the anti-hRSV F-protein antibody of the inventioncomprises a full tetrameric structure having two light chains and twoheavy chains, wherein each light chain comprises: a variable regioncomprising SEQ ID NO: 8 and a human kappa light chain constant domain(SEQ ID NO: 14); and each heavy chain comprises: a variable regioncomprising SEQ ID NO: 7 and a human IgG1 constant domain (SEQ ID NO:13).

In certain aspects of any of the above mentioned embodiments, theanti-hRSV F-protein antibody or antigen binding fragment thereof of theinvention can be conjugated to at least one prophylactic or therapeuticagent. In one embodiment, the therapeutic agent comprises a secondantibody or fragment thereof, an immunomodulator, a hormone, a cytotoxicagent, an enzyme, a radionuclide, a second antibody conjugated to atleast one immunomodulator, enzyme, radioactive label, hormone, antisenseoligonucleotide, or cytotoxic agent, or a combination thereof.

The invention also provides isolated polypeptides comprising the aminoacid sequence of any one of SEQ ID NOs: 1-8, 23 or 25, or a fragment ofany said sequences. In certain embodiments, the polypeptides comprisingheavy chain amino acid sequences do not comprise the amino acid sequenceof SEQ ID NO: 9.

The invention also provides isolated nucleic acids encoding any one ofthe anti-hRSV F-protein antibodies or antigen binding fragments of theinvention. In one embodiment, the invention provides isolated nucleicacids encoding any one of the polypeptides of SEQ ID NOs: 1-8, 23 or 25,wherein said polypeptides can optionally comprise a leader sequence. Incertain embodiments, the polypeptides comprising heavy chain amino acidsequences do not comprise the amino acid sequence of SEQ ID NO: 9. Theinvention also provides expression vectors comprising a nucleic acidencoding any one of the polypeptides of SEQ ID NOs: 1-8, 23 or 25(wherein said polypeptides can optionally comprise a leader sequence).In certain embodiments, the polypeptides comprising heavy chain aminoacid sequences do not comprise the amino acid sequence of SEQ ID NO: 9.These isolated nucleic acids and the expression vectors comprising themmay be used to express the antibodies of the invention or antigenbinding fragments thereof in recombinant host cells. Thus, the inventionalso provides host cells comprising isolated nucleic acids encoding anyone of the polypeptides of SEQ ID NOs: 1-8, 23 or 25 (wherein saidpolypeptides can optionally comprise a leader sequence). In certainembodiments, the polypeptides comprising heavy chain amino acidsequences do not comprise the amino acid sequence of SEQ ID NO: 9. Inone embodiment, the host cell is Chinese hamster ovary cell. In oneembodiment, the host cell is a yeast cell, for example a Pichia cell ora Pichia pastoris host cell.

The invention also provides pharmaceutical compositions comprising anantibody or antigen binding fragment of the invention and apharmaceutically acceptable carrier or diluent. In one embodiment, thepharmaceutically acceptable carrier or diluent is L-Histidine. In oneaspect of this embodiment, the antibody or antigen binding fragment isformulated in 10 mM L-Histidine, 7% (w/v) Sucrose, and 0.02% (w/v)polysorbate-80, pH 6.0. The antibody or antigen binding fragment istypically present at about 100 mg/mL in such a formulation.

In one embodiment, the present invention provides compositionscomprising an antibody or antigen binding fragment thereof of theinvention and comprising a further prophylactic or therapeutic agent. Inone embodiment, the further prophylactic or therapeutic agent isselected from the group consisting of: a second anti-hRSV antibody or anantigen binding fragment thereof. In one embodiment, the secondanti-hRSV antibody or antigen binding fragment of the inventioncomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3;(iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In certain embodiments, the heavy chain or heavy chain variableregion does not comprise the amino acid sequence of SEQ ID NO: 9.

The invention also provides a vessel or injection device comprising anyone of the anti-hRSV F-protein antibodies or antigen binding fragmentsof the invention. In one embodiment, the anti-hRSV F-protein antibody orantigen binding fragment of the invention comprises: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;(ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4;(v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6. In certainembodiments, the heavy chain or heavy chain variable region does notcomprise the amino acid sequence of SEQ ID NO: 9. In certainembodiments, the heavy chain comprises, consists essentially of, orconsists of, the amino acid sequence of SEQ ID NO: 23 and the lightchain comprises, consists essentially of, or consists of, the amino acidsequence of SEQ ID NO: 25.

The invention also provides a method of producing an anti-hRSV F-proteinantibody or antigen binding fragment of the invention comprising:culturing a host cell comprising a polynucleotide encoding a heavy chainand/or light chain of an antibody of the invention (or an antigenbinding fragment thereof) under conditions favorable to expression ofthe polynucleotide; and optionally, recovering the antibody or antigenbinding fragment from the host cell and/or culture medium. In oneembodiment, the polynucleotide encoding the heavy chain and thepolynucleotide encoding the light chain are in a single vector. Inanother embodiment, the polynucleotide encoding the heavy chain and thepolynucleotide encoding the light chain are in different vectors. In oneembodiment, the polynucleotide encoding the heavy chain and thepolynucleotide encoding the light chain encode an antibody or antigenbinding fragment comprising: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2;(iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO:4; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:5;and (vi) a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO:6. In certain embodiments, the heavy chain orheavy chain variable region does not comprise the amino acid sequence ofSEQ ID NO: 9. In certain embodiments, the heavy chain comprises,consists essentially of, or consists of, the amino acid sequence of SEQID NO: 23 and the light chain comprises, consists essentially of, orconsists of, the amino acid sequence of SEQ ID NO: 25.

The invention also provides a method of preventing or treating hRSVinfection in a subject in need thereof, comprising administering to thesubject an effective amount of an anti-hRSV F-protein antibody orantigen binding fragment of the invention, optionally in associationwith a further prophylactic or therapeutic agent or a therapeuticprocedure. In one embodiment, the subject being treated is a humansubject. In one embodiment, the further prophylactic or therapeuticagent is selected from the group consisting of: a second anti-hRSVantibody or an antigen binding fragment thereof, a nucleic acid encodingthe anti-RSV F antibody or antigen binding fragment, or an antibodyconjugate. In one embodiment, the anti-hRSV F-protein antibody orantigen binding fragment of the invention comprises: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;(ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4;(v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6. In certainembodiments, the heavy chain or heavy chain variable region does notcomprise the amino acid sequence of SEQ ID NO: 9. In certainembodiments, the heavy chain comprises, consists essentially of, orconsists of, the amino acid sequence of SEQ ID NO: 23 and the lightchain comprises, consists essentially of, or consists of, the amino acidsequence of SEQ ID NO: 25.

The invention also provides a method of preventing or treating hRSVinfection in a subject in need thereof, comprising administering to thesubject an effective amount of an anti-hRSV F-protein antibody orantigen binding fragment of the invention, optionally in combinationwith a further prophylactic or therapeutic agent or a therapeuticprocedure. In one embodiment, the anti-hRSV F-protein antibody orantigen binding fragment of the invention comprises: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;(ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4;(v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6. In certainembodiments, the heavy chain or heavy chain variable region does notcomprise the amino acid sequence of SEQ ID NO: 9. In certainembodiments, the heavy chain comprises, consists essentially of, orconsists of, the amino acid sequence of SEQ ID NO: 23 and the lightchain comprises, consists essentially of, or consists of, the amino acidsequence of SEQ ID NO: 25.

The invention also provides a vaccine, or immunogenic composition,comprising an antibody or antigen binding fragment of the invention. Inone embodiment, the anti-hRSV F-protein antibody or antigen bindingfragment of the invention comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 2; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5;and (vi) a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 6. In certain embodiments, the heavy chain orheavy chain variable region does not comprise the amino acid sequence ofSEQ ID NO: 9. In certain embodiments, the heavy chain comprises,consists essentially of, or consists of, the amino acid sequence of SEQID NO: 23 and the light chain comprises, consists essentially of, orconsists of, the amino acid sequence of SEQ ID NO: 25.

In one embodiment, the vaccine, or immunogenic composition, furthercomprises an antigen selected from RSV F protein and RSV G protein andfragments thereof.

The invention also provides a method for detecting the presence of RSVin a sample (by detecting F protein or a fragment thereof) comprisingcontacting the sample with an antibody or antigen binding fragmentthereof of the invention and detecting the presence of a complex betweenthe antibody or fragment and the peptide; wherein detection of thecomplex indicates the presence of RSV F protein. In one embodiment, theantibody or antigen binding fragment of the invention comprises: (i) aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 4; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6. In certainembodiments, the heavy chain or heavy chain variable region does notcomprise the amino acid sequence of SEQ ID NO: 9. In certainembodiments, the heavy chain comprises, consists essentially of, orconsists of, the amino acid sequence of SEQ ID NO: 23 and the lightchain comprises, consists essentially of, or consists of, the amino acidsequence of SEQ ID NO: 25.

The invention also provides a method of increasing the anti-hRSVactivity of an anti-hRSV F-protein antibody comprising: obtaining aparental anti-hRSV F-protein antibody and increasing the effectorfunction of the parental anti-hRSV F-protein antibody; wherein theactivity of the resulting anti-hRSV F-protein antibody is increased ascompared to the parental anti-hRSV F-protein antibody. As used herein, a“parental anti-antibody” refers to antibody having a wild-type Fc regionand/or wild type glycosylation (i.e., glycosylation pattern resultingfrom expression of the polypeptide in a non-engineered mammalian hostcell). The effector function of a parental antibody can be increased bymutating its Fc region or by altering its glycosylation, for example bymaking the antibody afucosylated (as discussed in further detail below).In one embodiment, the effector function of a parental anti-hRSVF-protein antibody is increased by making mutations in the Fc region ofthe parental anti-hRSV F-protein antibody. In another embodiment, theeffector function of a parental anti-hRSV F-protein antibody isincreased by removing the fucose residues from the antibody, orexpressing the antibody in a host cell that has been geneticallyengineered to remove the activity of the enzyme that adds fucose toglycoproteins.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B show binding curves (from ELISA) of human RSV antibodies D25,palivizumab, and RB1 to human RSV-F pre (A) and post (B) fusionproteins.

FIGS. 2A-B show neutralizing curves for human RSV antibodies in RSV ALong strain (A) and RSV B Washington strain (B).

FIGS. 3A-B show epitope mapping of RB1 by alanine scanning mutagenesisof Fusion F protein (A) and epitope mapped residues on Pre-Fusion Fcrystal structure (B).

FIGS. 4A-D show the efficacy of RB1 compared to D25 in lungs in a cottonrat challenge model of RSV A plotted against concentrations of antibody(A) and RSV B challenge plotted against concentrations of antibody (B)or viral particles (PFU/g) present in the tissues plotted against doseof antibody for RSV A challenge (C) and RSV B challenge (D).

FIGS. 5A-D show the efficacy of RB1 compared to D25 in nose in a cottonrat challenge model of RSV A plotted against concentrations of antibody(A) and RSV B challenge plotted against concentrations of antibody (B)or viral particles (PFU/g) present in the tissues plotted against doseof antibody for RSV A challenge (C) and RSV B challenge (D).

FIG. 6 shows a binding curve (from ELISA) of human RSV antibody RB1+YTEto human RSV A F protein.

FIG. 7 shows pharmacokinetic properties in Rhesus of RB1−YTE (RB1+YTE)vs. motavizumab having the YTE mutation set.

DETAILED DESCRIPTION Abbreviations

Throughout the detailed description and examples of the invention thefollowing abbreviations will be used:

-   -   ADCC Antibody-dependent cellular cytotoxicity    -   CDC Complement-dependent cytotoxicity    -   CDR Complementarity determining region in the immunoglobulin        variable regions, defined using the Kabat numbering system    -   CHO Chinese hamster ovary    -   ELISA Enzyme-linked immunosorbant assay    -   FR Antibody framework region: the immunoglobulin variable        regions excluding the CDR regions    -   HRP Horseradish peroxidase    -   IC50 concentration resulting in 50% inhibition    -   IgG Immunoglobulin G    -   Kabat An immunoglobulin alignment and numbering system pioneered        by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological        Interest, 5th Ed. Public Health Service, National Institutes of        Health, Bethesda, Md.)    -   mAb or Mab or MAb Monoclonal antibody    -   V region The segment of IgG chains which is variable in sequence        between different antibodies. It extends to Kabat residue 109 in        the light chain and 113 in the heavy chain.    -   VH Immunoglobulin heavy chain variable region    -   VK Immunoglobulin kappa light chain variable region

Definitions

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding compound, or by another cell.

“RSV disease” means any disease caused, directly or indirectly, by aninfection with Respiratory Syncytial Virus (RSV) as well as diseases orconditions which predispose a patient to infection by RSV. Examples ofdiseases falling into the former category include pneumonia andbronchiolitis. Diseases and conditions in the latter category (i.e.,those which place the patient at risk of severe RSV infection) includecystic fibrosis, congenital heart disease, cancer, age relatedimmunosuppression, transplant recipients and, generally, any conditionthat causes a state of immunosuppression or decreased function of theimmune system such as post- operative organ transplantation regimens orpremature birth.

“Treat” or “treating” means to administer a therapeutic agent, such as acomposition containing any of the antibodies or antigen-bindingfragments of the present invention, internally or externally to asubject or patient having one or more disease symptoms, or beingsuspected of having a disease, for which the agent has therapeuticactivity. Typically, the agent is administered in an amount effective toalleviate one or more disease symptoms in the treated subject orpopulation, whether by inducing the regression of or inhibiting theprogression of such symptom(s) by any clinically measurable degree. Theamount of a therapeutic agent that is effective to alleviate anyparticular disease symptom may vary according to factors such as thedisease state, age, and weight of the patient, and the ability of thedrug to elicit a desired response in the subject. Whether a diseasesymptom has been alleviated can be assessed by any clinical measurementtypically used by physicians or other skilled healthcare providers toassess the severity or progression status of that symptom. Treatmentwith anti-RSV antibodies could also combined with other interventions(antibodies, nucleic acids, vaccines and small molecule compounds) totreat other respiratory pathogens.

“Prevent” or “preventing” means to administer a prophylactic agent, suchas a composition containing any of the antibodies or antigen-bindingfragments of the present invention, internally or externally to asubject or patient at risk of becoming infected by hRSV, for which theagent has prophylactic activity. Preventing includes reducing thelikelihood or severity of a subsequent RSV infection, amelioratingsymptoms associated with lower respiratory tract infection (LRI) uponRSV infection, and inducing immunity to protect against RSV infection.Typically, the agent is administered in an amount effective toneutralize RSV in the lungs and/or the nose in order block infection.The amount of a prophylactic agent that is effective to ameliorate anyparticular disease symptom may vary according to factors such as theage, and weight of the patient, and the ability of the agent to elicit adesired response in the subject. Whether a disease symptom has beenameliorated can be assessed by any clinical measurement typically usedby physicians or other skilled healthcare providers to assess theseverity or progression status of that symptom or in certain instanceswill ameliorate the need for hospitalization.

hRSV F Protein

Human RSV F protein is synthesized as a metastable trimeric precursor(F0) that is proteolytically cleaved into the covalently associated F1and F2 subunits. Atomic structures of F trimers in the prefusion formhave been determined for PIVS and RSV members of paramyxoviridae family.See McLellan et al., 2011, J Virol. 85:7788-7796 (RSV) and Welch et al.,2012, Proc Natl Acad Sci 109:16672-16677 (PIV). Prefusion F has a shortC-terminal cytoplasmic tail, a single transmembrane domain, a helicalstalk, and a globular head domain. Atomic structures of NDV, hPIV3, andRSV F in the postfusion form reveal that a large refolding event occursto convert prefusion F to postfusion F in which part of the globularhead domain rearranges to form a six helix bundle. These structures,along with peptide inhibitory data, suggest a model for F mediatedmembrane fusion where, upon activation, F1/F2 rearranges to insert ahydrophobic fusion peptide from the N-terminus of Fl into the targetcell membrane forming a pre-hairpin intermediate. This relativelyextended structure tethers the virus to the cell membrane and collapsesto form the stable six-helix bundle of the postfusion structure. Thetransition from the metastable prefusion, to the prehairpinintermediate, to the postfusion conformation proceeds down an energygradient with the postfusion form representing the most stable state,and the energy released during F refolding is coupled with membranefusion.

The term hRSV F protein includes human RSV F protein as well asfragments thereof such as the mature fragment thereof lacking the signalpeptide. In an embodiment of the invention, the amino acid sequence ofhuman RSV F protein comprises the amino acid sequence disclosed inGenbank Accession Number AAR14266 (hRSV B strain 9320).

Anti-hRSV Antibodies and Antigen-Binding Fragments Thereof

The present invention provides antibodies or antigen-binding fragmentsthereof that bind human RSV F protein, preferably from both RSV Astrains and B strains, that bind both the pre-fusion F protein and thepost-fusion F protein, and uses of such antibodies or fragments. In someembodiments, the anti-RSV F-protein antibodies are isolated. Theantibodies described herein bind to an epitope at site IV of the Fprotein. In any of the embodiments of the invention described herein, incertain embodiments, the heavy chain or heavy chain variable region doesnot comprise the amino acid sequence of SEQ ID NO: 9 and/or the lightchain or light chain variable region does not comprise the amino acidsequence of SEQ ID NO: 8. In certain embodiments, the heavy chaincomprises, consists essentially of, or consists of, the amino acidsequence of SEQ ID NO: 23 and the light chain comprises, consistsessentially of, or consists of, the amino acid sequence of SEQ ID NO:25.

In preferred embodiments, the anti-RSV F-protein antibodies are fullyhuman. A major advantage of the monoclonal antibodies of the inventionderives from the fact that they include human CDR3 sequences and, insome embodiments, may be entirely human monoclonal antibodies. Hence invivo use of the fully human monoclonal antibodies of the invention forimmunoprophylaxis and immunotherapy of RSV disease greatly reduces theproblem of host immune response to passively administered antibodies.This problem is commonly encountered when the prior art monoclonalantibodies of xenogeneic or chimeric derivation are utilized. A secondimportant aspect of this advantage is the potential safety of thesehuman monoclonal antibodies for gene therapy applications, in whichexpression of xenogeneic or chimeric proteins containing non-humansequences cannot be terminated.

As used herein, an anti-RSV F-protein antibody or antigen-bindingfragment thereof refers to an antibody or antigen-binding fragmentthereof that specifically binds to human RSV F protein. An antibody orantigen-binding fragment thereof that “specifically binds to human RSV”is an antibody or antigen-binding fragment thereof that binds to thepre-fusion or post-fusion human RSV F protein with a Kd of about 1 nM ora higher affinity (e.g., 1 nM-2 pM, 1 nM, 100 pM, 10 pM or 2 pM), butdoes not bind to other proteins lacking RSV F protein sequences. In oneembodiment, the antibody of the invention which specifically binds tohuman RSV F protein is also cross-reactive with bovine RSV F protein. Asused herein “cross-reactivity” refers to the ability of an antibody toreact with a homologous protein from other species. Whether an antibodyspecifically binds to human RSV F protein can be determined using anyassay known in the art. Examples of assays known in the art todetermining binding affinity include surface plasmon resonance (e.g.,BIACORE) or a similar technique (e.g. KinExa or OCTET).

The present invention includes anti-hRSV F-protein antibodies andmethods of use thereof. As used herein, the term “antibody” refers toany form of antibody that exhibits the desired biological activity.Thus, it is used in the broadest sense and specifically covers, but isnot limited to, monoclonal antibodies (including full length monoclonalantibodies comprising two light chains and two heavy chains), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies),humanized antibodies, fully human antibodies, and chimeric antibodies.

The present invention includes anti-hRSV F-protein antigen-bindingfragments and methods of use thereof. As used herein, unless otherwiseindicated, “antibody fragment” or “antigen-binding fragment” refers toantigen-binding fragments of antibodies, i.e. antibody fragments thatretain the ability to bind specifically to the antigen bound by thefull-length antibody, e.g., fragments that retain one or more CDRregions. Examples of antigen-binding fragments include, but are notlimited to, Fab, Fab′, F(ab′)₂, and FIT fragments; diabodies; linearantibodies; single-chain antibody molecules, e.g., sc-Fv; andmultispecific antibodies formed from antibody fragments.

The present invention includes anti-RSV F-protein Fab fragments andmethods of use thereof. A “Fab fragment” is comprised of one light chainand the C_(H)1 and variable regions of one heavy chain. The heavy chainof a Fab molecule cannot form a disulfide bond with another heavy chainmolecule. An “Fab fragment” can be the product of papain cleavage of anantibody.

The present invention includes anti-RSV F-protein antibodies andantigen-binding fragments thereof which comprise an Fc region andmethods of use thereof. An “Fc” region contains two heavy chainfragments comprising the C_(H)1 and C_(H)2 domains of an antibody. Thetwo heavy chain fragments are held together by two or more disulfidebonds and by hydrophobic interactions of the C_(H)3 domains.

The present invention includes anti-RSV F-protein Fab′ fragments andmethods of use thereof. A “Fab′ fragment” contains one light chain and aportion or fragment of one heavy chain that contains the V_(H) domainand the C_(H)1 domain and also the region between the C_(H)1 and C_(H)2domains, such that an interchain disulfide bond can be formed betweenthe two heavy chains of two Fab′ fragments to form a F(ab′)₂ molecule.

The present invention includes anti-RSV F-protein F(ab′)₂ fragments andmethods of use thereof. A “F(ab′)₂ fragment” contains two light chainsand two heavy chains containing a portion of the constant region betweenthe C_(H1) and C_(H2) domains, such that an interchain disulfide bond isformed between the two heavy chains. A F(ab′) 2 fragment thus iscomposed of two Fab′ fragments that are held together by a disulfidebond between the two heavy chains. An “F(ab′)₂ fragment” can be theproduct of pepsin cleavage of an antibody.

The present invention includes anti-RSV F-protein Fv fragments andmethods of use thereof. The “Fv region” comprises the variable regionsfrom both the heavy and light chains, but lacks the constant regions.

The present invention includes anti-RSV F-protein scFv fragments andmethods of use thereof. The term “single-chain Fv” or “scFv” antibodyrefers to antibody fragments comprising the V_(H) and V_(L) domains ofan antibody, wherein these domains are present in a single polypeptidechain. Generally, the Fv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains which enables the scFv toform the desired structure for antigen-binding. For a review of scFv,see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.See also, International Patent Application Publication No. WO 88/01649and U.S. Pat. Nos. 4,946,778 and 5,260,203.

The present invention includes anti-RSV F-protein domain antibodies andmethods of use thereof. A “domain antibody” is an immunologicallyfunctional immunoglobulin fragment containing only the variable regionof a heavy chain or the variable region of a light chain. In someinstances, two or more V_(H) regions are covalently joined with apeptide linker to create a bivalent domain antibody. The two V_(H)regions of a bivalent domain antibody may target the same or differentantigens.

The present invention includes anti-RSV F-protein bivalent antibodiesand methods of use thereof. A “bivalent antibody” comprises twoantigen-binding sites. In some instances, the two binding sites have thesame antigen specificities. However, bivalent antibodies may bebispecific (see below).

The present invention includes anti-RSV F-protein diabodies and methodsof use thereof. As used herein, the term “diabodies” refers to smallantibody fragments with two antigen-binding sites, which fragmentscomprise a heavy chain variable domain (V_(H)) connected to a lightchain variable domain (V_(L)) in the same polypeptide chain (V_(H)-V_(L)or V_(L)-V_(H)). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, e.g., EP404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci.USA 90: 6444-6448. For a review of engineered antibody variantsgenerally see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

Typically, an antibody or antigen-binding fragment of the inventionwhich is modified in some way retains at least 10% of its bindingactivity (when compared to the parental antibody) when that activity isexpressed on a molar basis. Preferably, an antibody or antigen-bindingfragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95%or 100% or more of the RSV F-protein binding affinity as the parentalantibody. It is also intended that an antibody or antigen-bindingfragment of the invention can include conservative or non-conservativeamino acid substitutions (referred to as “conservative variants” or“function conserved variants” of the antibody) that do not substantiallyalter its biologic activity.

The present invention includes isolated anti-hRSV F-protein antibodiesand antigen-binding fragments thereof and methods of use thereof.“Isolated” antibodies or antigen-binding fragments thereof are at leastpartially free of other biological molecules from the cells or cellcultures in which they are produced. Such biological molecules includenucleic acids, proteins, lipids, carbohydrates, or other material suchas cellular debris and growth medium. An isolated antibody orantigen-binding fragment may further be at least partially free ofexpression system components such as biological molecules from a hostcell or of the growth medium thereof. Generally, the term “isolated” isnot intended to refer to a complete absence of such biological moleculesor to an absence of water, buffers, or salts or to components of apharmaceutical formulation that includes the antibodies or fragments.

The present invention includes monoclonal anti-hRSV F-protein antibodiesand antigen-binding fragments thereof as well as monoclonal antibodycompositions comprising a plurality of isolated monoclonal antibodies.The term “monoclonal antibody”, as used herein, refers to a populationof substantially homogeneous antibodies, i.e., the antibody moleculescomprising the population are identical in amino acid sequence exceptfor possible naturally occurring mutations that may be present in minoramounts. In contrast, conventional (polyclonal) antibody preparationstypically include a multitude of different antibodies having differentamino acid sequences in their variable domains, particularly their CDRsthat are often specific for different epitopes. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by recombinant DNA methods (see,e.g., U.S. Pat. No. 4,816,567).

In general, the basic (or “full-length”) antibody structural unitcomprises a tetramer. Each tetramer includes two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region or domain of about 100 to 110 or moreamino acids primarily responsible for antigen recognition. Thecarboxy-terminal portion of the heavy chain may define a constant regionor domain primarily responsible for effector function. Typically, humanlight chains are classified as kappa and lambda light chains.Furthermore, human heavy chains are typically classified as mu, delta,gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD,IgG, IgA, and IgE, respectively. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. See generally, Fundamental Immunology Ch. 7(Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989). In the context of anantibody or antigen binding fragment thereof, the terms domain andregion can be used interchangeably, where appropriate.

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, in general, an intact antibody has two bindingsites. Except in bifunctional or bispecific antibodies, the two bindingsites are, in general, the same.

Typically, the variable domains of both the heavy and light chainscomprise three hypervariable regions, also called complementaritydetermining regions (CDRs), located within relatively conservedframework regions (FR). The CDRs are usually aligned by the frameworkregions, enabling binding to a specific epitope. In general, fromN-terminal to C-terminal, both light and heavy chains variable domainscomprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment ofamino acids to each domain is, generally, in accordance with thedefinitions of Sequences of Proteins of Immunological Interest, Kabat,et al.; National Institutes of Health, Bethesda, Md.; 5^(th) ed.; NIHPubl. No. 91-3242 (1991); Kabat, 1978, Adv. Prot. Chem. 32:1-75; Kabat,et al., 1977, J Biol. Chem. 252:6609-6616; Chothia et al., 1987, J Mol.Biol. 196:901-917 or Chothia et al., 1989, Nature 342:878-883.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody or antigen-binding fragment thereof that areresponsible for antigen-binding. The hypervariable region comprisesamino acid residues from a “complementarity determining region” or “CDR”(i.e. CDRL1, CDRL2 and CDRL3 in the light chain variable domain andCDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). See Kabat etal. (1991) Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(defining the CDR regions of an antibody by sequence); see also Chothiaand Lesk, 1987, J Mol. Biol. 196: 901-917 (defining the CDR regions ofan antibody by structure). As used herein, the term “framework” or “FR”residues refers to those variable domain residues other than thehypervariable region residues defined herein as CDR residues.

“Isolated nucleic acid molecule” or “isolated polynucleotide” means aDNA or RNA of genomic, mRNA, cDNA, or synthetic origin or somecombination thereof which is not associated with all or a portion of apolynucleotide in which the isolated polynucleotide is found in nature,or is linked to a polynucleotide to which it is not linked in nature.For purposes of this disclosure, it should be understood that “a nucleicacid molecule comprising” a particular nucleotide sequence does notencompass intact chromosomes. Isolated nucleic acid molecules“comprising” specified nucleic acid sequences may include, in additionto the specified sequences, coding sequences for up to ten or even up totwenty or more other proteins or portions or fragments thereof, or mayinclude operably linked regulatory sequences that control expression ofthe coding region of the recited nucleic acid sequences, and/or mayinclude vector sequences.

The phrase “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to use promoters,polyadenylation signals, and enhancers.

A nucleic acid or polynucleotide is “operably linked” when it is placedinto a functional relationship with another nucleic acid sequence. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, but not always, “operably linked” means that the DNAsequences being linked are contiguous, and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that not all progeny willhave precisely identical DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “germline sequence” refers to a sequence of unrearrangedimmunoglobulin DNA sequences. Any suitable source of unrearrangedimmunoglobulin sequences may be used. Human germline sequences may beobtained, for example, from JOINSOLVER germline databases on the websitefor the National Institute of Arthritis and Musculoskeletal and SkinDiseases of the United States National Institutes of Health. Mousegermline sequences may be obtained, for example, as described inGiudicelli et al., 2005, Nucleic Acids Res. 33 :D256-D261.

Physical and Functional Properties of the Exemplary Anti-RSV F-ProteinAntibodies

The present invention provides anti-hRSV F-protein antibodies andantigen-binding fragments thereof having specified structural andfunctional features, and methods of use of the antibodies orantigen-binding fragments thereof in the treatment or prevention ofdiseases/conditions associated with RSV infection.

An “anti-RSV F-protein antibody or antigen-binding fragment thereof ofthe present invention” includes: any antibody or antigen-bindingfragment thereof that is discussed herein (e.g., RB1) or a variantthereof (e.g., sequence variant or functional variant); any antibody orantigen-binding fragment comprising any one or more of the CDRs setforth in Table 7; any antibody or antigen-binding fragment that binds tothe same epitope in human RSV F-protein as the antibodies discussedherein (e.g., RB1); and any antibody or antigen-binding fragment thatcross-blocks (partially or fully) or is cross-blocked (partially orfully) by an antibody discussed herein (e.g., RB1) for RSV binding.

Cross-blocking antibodies and antigen-binding fragments thereof can beidentified based on their ability to cross-compete with an antibody ofthe invention in standard binding assays (e.g., BIACore, ELISA, flowcytometry). For example, standard ELISA assays can be used in which arecombinant RSV F protein protein is immobilized on the plate, one ofthe antibodies is fluorescently labeled and the ability of non-labeledantibodies to compete off the binding of the labeled antibody isevaluated. Additionally or alternatively, BIAcore analysis can be usedto assess the ability of the antibodies to cross-compete. The ability ofa test antibody to inhibit the binding of another antibody (for example,antibody D25) to RSV F-protein demonstrates that the test antibody cancompete with another antibody (e.g., D25) for binding to RSV F proteinand thus, may, in some cases, bind to the same epitope on RSV F proteinas antibody D25 to an overlapping epitope.

As stated above, antibodies and fragments thereof that bind to the sameepitope as any of the anti-RSV F-protein antibodies or antigen-bindingfragments thereof of the present invention also form part of the presentinvention. Further, in certain embodiments, antibodies that bind to anepitope that overlaps with the epitope bound by any of the anti-RSVF-protein antibodies of the invention also form part of the presentinvention. There are several methods available for mapping antibodyepitopes on target antigens, including: H/D-Ex Mass spec, X-raycrystallography, pepscan analysis and site directed mutagenesis. Forexample, HDX (Hydrogen Deuterium Exchange) coupled with proteolysis andmass spectrometry can be used to determine the epitope of an antibody ona specific antigen Y. HDX-MS relies on the accurate measurement andcomparison of the degree of deuterium incorporation by an antigen whenincubated in D₂O on its own and in presence of its antibody at varioustime intervals. Deuterium is exchanged with hydrogen on the amidebackbone of the proteins in exposed areas whereas regions of the antigenbound to the antibody will be protected and will show less or noexchange after analysis by LC-MS/MS of proteolytic fragments.

Examples of the immunoglobulin chains of anti-RSV F-protein antibodiesof the invention as well as their CDRs include, but are not limitedthose disclosed in Table 7 (SEQ ID NOs: 1-8, 23 and 25). The presentinvention includes any polypeptide comprising, consisting essentiallyof, or consisting of the amino acid sequences of SEQ ID NOs: 1-8, 23,and 25, and recombinant nucleotides encoding such polypeptides.

The scope of the present invention includes isolated anti-hRSV F-proteinantibodies and antigen-binding fragments thereof, comprising a variantof an immunoglobulin chain set forth herein, e.g., any of SEQ ID NOs: 7,8; wherein the variant exhibits one or more of the following properties:(i) binds to human RSV pre-fusion F protein with a Kd value of about1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmon resonance(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); or (ii)binds to human RSV post-fusion F protein with a Kd value of about 1×10⁻⁹M to about 1×10⁻¹¹ M as determined by surface plasmon resonance (e.g.,BIACORE) or a similar technique (e.g. KinExa or OCTET). In certainembodiments, the heavy chain or heavy chain variable region does notcomprise the amino acid sequence of SEQ ID NO: 9.

In certain embodiments, the invention provides antibodies orantigen-binding fragment thereof that binds human hRSV F-protein and hasV_(L) domains and V_(H) domains with at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity with SEQ ID NOs: 8 (V_(L)) and 7(V_(H)); wherein the variant exhibits the desired binding andproperties, e.g., (i) binds to human RSV pre-fusion F protein with a Kdvalue of about 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa orOCTET); or (ii) binds to human RSV post-fusion F protein with a Kd valueof about 1×10⁻⁹ M to about 1×10⁻¹¹ M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).In certain embodiments, the heavy chain or heavy chain variable regiondoes not comprise the amino acid sequence of SEQ ID NO: 9.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids in a protein with other amino acidshaving similar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering thebiological activity of the protein. Those of skill in this art recognizethat, in general, single amino acid substitutions in non-essentialregions of a polypeptide do not substantially alter biological activity(see, e.g., Watson et al. (1987) Molecular Biology of the Gene, TheBenjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,substitutions of structurally or functionally similar amino acids areless likely to disrupt biological activity. Exemplary conservativesubstitutions are set forth in Table 1.

TABLE 1 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

Function-conservative variants of the antibodies of the invention arealso contemplated by the present invention. “Function-conservativevariants,” as used herein, refers to antibodies or fragments in whichone or more amino acid residues have been changed without altering adesired property, such an antigen affinity and/or specificity. Suchvariants include, but are not limited to, replacement of an amino acidwith one having similar properties, such as the conservative amino acidsubstitutions of Table 1. Also provided are isolated polypeptidescomprising the V_(L) domains of the anti-hRSV F-protein antibodies ofthe invention (e.g., SEQ ID NO: 8), and isolated polypeptides comprisingthe V_(H) domains (e.g., SEQ ID NO: 7) of the anti-hRSV antibodies ofthe invention having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore amino acid substitutions, which may occur exclusively in theframework region or of which one or more may be located in one or moreCDRs . In certain embodiments, the heavy chain or heavy chain variableregion does not comprise the amino acid sequence of SEQ ID NO: 9.

In another embodiment, provided is an antibody or antigen-bindingfragment thereof that binds hRSV F-protein and has V_(L) domains andV_(H) domains with at least 99% 98%, 97%, 96%, 95%, 90%, 85%, 80% or 75%sequence identity to one or more of the V_(L) domains or V_(H) domainsdescribed herein, and exhibits specific binding to hRSV F-protein. Inanother embodiment the binding antibody or antigen-binding fragmentthereof of the present invention comprises V_(L) and V_(H) domains (withand without signal sequence) having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 or more amino acid substitutions, which may occur exclusivelyin the framework region or of which one or more may be located in one ormore CDRs, and exhibits specific binding to hRSV F-protein. In certainembodiments, the heavy chain or heavy chain variable region does notcomprise the amino acid sequence of SEQ ID NO: 9.

Polynucleotides and Polypeptides

The present invention further comprises the polynucleotides encoding anyof the polypeptides or immunoglobulin chains of anti-hRSV F-proteinantibodies and antigen-binding fragments thereof of the invention. Inone embodiment, the isolated polynucleotide encodes an antibody orantigen-binding fragment thereof comprising at least one matureimmunoglobulin light chain variable (V_(L)) domain according to theinvention and/or at least one mature immunoglobulin heavy chain variable(V_(H)) domain according to the invention. In some embodiments theisolated polynucleotide encodes both a light chain and a heavy chain ona single polynucleotide molecule, and in other embodiments the light andheavy chains are encoded on separate polynucleotide molecules. Inanother embodiment the polynucleotides further encodes a signalsequence. For example, the present invention includes thepolynucleotides encoding the amino acids described in SEQ ID NOs: 1-8,23 and 25, as well as polynucleotides which hybridize thereto and, also,any polypeptide encoded by such a hybridizing polynucleotide. In oneembodiment, the invention comprises a nucleic acid sequence comprising,consisting essentially of, or consisting of SEQ ID NO: 15 (variableheavy chain) or SEQ ID NO: 16 (variable light chain). In certainembodiments, codon optimization can be used to enhance a property of thenucleic acid, e.g., expression in a certain host. In one embodiment, theinvention comprises a nucleic acid sequence comprising, consistingessentially of, or consisting of SEQ ID NO: 17 (codon optimized variableheavy) or SEQ ID NO: 18 (codon optimized variable light). In certainembodiments, a leader sequence can be used. In one embodiment, theinvention comprises a nucleic acid sequence comprising, consistingessentially of, or consisting of SEQ ID NO: 19 (leader sequence andheavy chain) or SEQ ID NO: 20 (leader sequence and light chain)connected with the heavy chain or light chain to give SEQ ID NO: 21 orSEQ ID NO: 22, respectively.

In general, the polynucleotides hybridize under low, moderate or highstringency conditions, and encode antibodies or antigen-bindingfragments thereof that maintain the ability to bind to hRSV F-protein. Afirst polynucleotide molecule is “hybridizable” to a secondpolynucleotide molecule when a single stranded form of the firstpolynucleotide molecule can anneal to the second polynucleotide moleculeunder the appropriate conditions of temperature and solution ionicstrength (see Sambrook, et al., supra). The conditions of temperatureand ionic strength determine the “stringency” of the hybridization.Typical low stringency hybridization conditions include 55° C., 5×SSC,0.1% SDS and no formamide; or 30% formamide, 5×SSC, 0.5% SDS at 42° C.Typical moderate stringency hybridization conditions are 40% formamide,with 5× or 6×SSC and 0.1% SDS at 42° C. High stringency hybridizationconditions are 50% formamide, 5× or 6×SSC at 42° C. or, optionally, at ahigher temperature (e.g., 57° C., 59° C., 60° C., 62° C., 63° C., 65° C.or 68° C.). In general, SSC is 0.15M NaCl and 0.015M Na-citrate.Hybridization requires that the two polynucleotides containcomplementary sequences, although, depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing polynucleotides depends on the length of thepolynucleotides and the degree of complementation, variables well knownin the art. The greater the degree of similarity or homology between twonucleotide sequences, the higher the stringency under which the nucleicacids may hybridize. For hybrids of greater than 100 nucleotides inlength, equations for calculating the melting temperature have beenderived (see Sambrook et al., supra, 9.50-9.51). For hybridization withshorter polynucleotides, e.g., oligonucleotides, the position ofmismatches becomes more important, and the length of the oligonucleotidedetermines its specificity (see Sambrook, et al., supra, 11.7-11.8).

In one embodiment, the invention comprises an isolated polynucleotideencoding an antibody heavy variable (V_(H)) domain or an antigen-bindingfragment thereof comprising CDR-H1 (SEQ ID NO: 1), CDR-H2 (SEQ ID NO: 2)and CDR-H3 (SEQ ID NO: 3).

In one embodiment, the invention comprises an isolated polynucleotideencoding an antibody light chain variable (V_(L)) domain or anantigen-binding fragment thereof comprising CDR-L1 (SEQ ID NO: 4),CDR-L2 (SEQ ID NO: 5) and CDR-L3 (SEQ ID NO: 6).

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(H)) domain of SEQID NO: 7 or a heavy chain of SEQ ID NO: 23.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(L)) domain of SEQID NO: 8 or a light chain of SEQ ID NO: 25.

This present invention also provides vectors, e.g., expression vectors,such as plasmids, comprising the isolated polynucleotides of theinvention, wherein the polynucleotide is operably linked to controlsequences that are recognized by a host cell when the host cell istransfected with the vector. Also provided are host cells comprising avector of the present invention and methods for producing the antibodyor antigen-binding fragment thereof or polypeptide disclosed hereincomprising culturing a host cell harboring an expression vector or anucleic acid encoding the immunoglobulin chains of the antibody orantigen-binding fragment thereof in culture medium, and isolating theantigen or antigen-binding fragment thereof from the host cell orculture medium.

Also included in the present invention are polypeptides, e.g.,immunoglobulin polypeptides, comprising amino acid sequences that are atleast about 75% identical, 80% identical, more preferably at least about90% identical and most preferably at least about 95% identical (e.g.,95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequences of theantibodies provided herein when the comparison is performed by a BLASTalgorithm wherein the parameters of the algorithm are selected to givethe largest match between the respective sequences over the entirelength of the respective reference sequences (e.g. expect threshold: 10;word size: 3; max matches in a query range: 0; BLOSUM 62 matrix; gapcosts: existence 11, extension 1; conditional compositional score matrixadjustment).

Sequence identity refers to the degree to which the amino acids of twopolypeptides are the same at equivalent positions when the two sequencesare optimally aligned.

The following references relate to BLAST algorithms often used forsequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J.272(20): 5101-5109; Altschul, S. F., et al., (1990) J. Mol. Biol.215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden,T. L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F., etal., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997)Genome Res. 7:649-656; Wootton, J. C., et al., (1993) Comput. Chem.17:149-163; Hancock, J. M. et al., (1994) Comput. Appl. Biosci.10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “A model ofevolutionary change in proteins.” in Atlas of Protein Sequence andStructure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352,Natl. Biomed. Res. Found., Washington, DC; Schwartz, R. M., et al.,“Matrices for detecting distant relationships.” in Atlas of ProteinSequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff (ed.),pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, S. F.,(1991) J. Mol. Biol. 219:555-565; States, D. J., et al., (1991) Methods3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol.36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl.Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.22:2022-2039; and Altschul, S. F. “Evaluating the statisticalsignificance of multiple distinct local alignments.” in Theoretical andComputational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, New York.

Binding Affinity

By way of example, and not limitation, the antibodies andantigen-binding fragments disclosed herein may bind human RSV pre-fusionF protein or post-fusion F protein with a K_(D) value of at least about1×10⁻⁹ M (i.e, a K_(D) value of 1×10⁻⁹ M or lower) as determined bysurface plasmon resonance (e.g., BIACORE) or a similar technique (e.g.KinExa or OCTET). In one embodiment, the antibodies and antigen-bindingfragments disclosed herein may bind human RSV pre-fusion F protein orpost-fusion F protein with a K_(D) value of at least about 1×10⁻⁹ M toabout 1×10⁻¹² M as determined by surface plasmon resonance (e.g.,BIACORE) or a similar technique (e.g. KinExa or OCTET). In oneembodiment, the antibodies and antigen-binding fragments disclosedherein may bind human RSV pre-fusion F protein or post-fusion F proteinwith a K_(D) value of at about 1×10⁻⁹ M to about 1×10⁻¹² M as determinedby surface plasmon resonance (e.g., BIACORE) or a similar technique(e.g. KinExa or OCTET). In one embodiment, the antibodies andantigen-binding fragments disclosed herein may bind human RSV pre-fusionF protein or post-fusion F protein with a K_(D) value of at least about100 pM (i.e, a K_(D) value of about 100 pM or lower) as determined byBIACORE or a similar technique. In one embodiment, the antibodies andantigen-binding fragments disclosed herein may bind human RSV pre-fusionF protein or post-fusion F protein with a K_(D) value of at least about10 pM (i.e., a K_(D) value of about 10 pm lower) as determined byBIACORE or a similar technique. In one embodiment, the antibodies andantigen-binding fragments of the invention may bind to human RSVpre-fusion F protein or post-fusion F protein with a K_(D) of about 1 pMto about 100 pM as determined by BIACORE or a similar technique.

Methods of Making Antibodies and Antigen-Binding Fragments Thereof

The present invention includes methods for making an anti-hRSV F-proteinantibody or antigen-binding fragment thereof of the present inventioncomprising culturing a cell line that expresses the antibody or fragmentunder conditions favorable to such expression and, optionally, isolatingthe antibody or fragment from the cells and/or the growth medium (e.g.cell culture medium).

The anti-hRSV F-protein antibodies disclosed herein may also be producedrecombinantly (e.g., in an E. coli/T7 expression system, a mammaliancell expression system or a lower eukaryote expression system). In thisembodiment, nucleic acids encoding the antibody immunoglobulin moleculesof the invention (e.g., V_(H) or V_(L)) may be inserted into a pET-basedplasmid and expressed in the E. coli/T7 system. For example, the presentinvention includes methods for expressing an antibody or antigen-bindingfragment thereof or immunoglobulin chain thereof in a host cell (e.g.,bacterial host cell such as E. coli such as BL21 or BL21DE3) comprisingexpressing T7 RNA polymerase in the cell which also includes apolynucleotide encoding an immunoglobulin chain that is operably linkedto a T7 promoter. For example, in an embodiment of the invention, abacterial host cell, such as a E. coli, includes a polynucleotideencoding the T7 RNA polymerase gene operably linked to a lac promoterand expression of the polymerase and the chain is induced by incubationof the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside).

There are several methods by which to produce recombinant antibodieswhich are known in the art. One example of a method for recombinantproduction of antibodies is disclosed in U.S. Pat. No. 4,816,567.

Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,biolistic injection and direct microinjection of the DNA into nuclei. Inaddition, nucleic acid molecules may be introduced into mammalian cellsby viral vectors. Methods of transforming cells are well known in theart. See, for example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461and 4,959,455.

Thus, the present invention includes recombinant methods for making ananti-hRSV antibody or antigen-binding fragment thereof of the presentinvention, or an immunoglobulin chain thereof, comprising introducing apolynucleotide encoding one or more immunoglobulin chains of theantibody or fragment (e.g., heavy and/or light immunoglobulin chain);culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) undercondition favorable to such expression and, optionally, isolating theantibody or fragment or chain from the host cell and/or medium in whichthe host cell is grown.

Anti-hRSV F-protein antibodies can also be synthesized by any of themethods set forth in U.S. Pat. No. 6,331,415.

Eukaryotic and prokaryotic host cells, including mammalian cells ashosts for expression of the antibodies or fragments or immunoglobulinchains disclosed herein are well known in the art and include manyimmortalized cell lines available from the American Type CultureCollection (ATCC). These include, inter alia, Chinese hamster ovary(CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number ofother cell lines. Mammalian host cells include human, mouse, rat, dog,monkey, pig, goat, bovine, horse and hamster cells. Cell lines ofparticular preference are selected through determining which cell lineshave high expression levels. Other cell lines that may be used areinsect cell lines, such as Sf9 cells, amphibian cells, bacterial cells,plant cells and fungal cells. Fungal cells include yeast and filamentousfungus cells including, for example, Pichia pastoris, Pichia finlandica,Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichiaminuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum,Physcomitrella patens and Neurospora crassa. Pichia sp., anySaccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candidaalbicans, any Aspergillus sp., Trichoderma reesei, Chrysosporiumlucknowense, any Fusarium sp., Yarrowia hpolytica, and Neurosporacrassa. When recombinant expression vectors encoding the heavy chain orantigen-binding portion or fragment thereof, the light chain and/orantigen-binding fragment thereof are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody orfragment or chain in the host cells or secretion of the into the culturemedium in which the host cells are grown.

Antibodies and antigen-binding fragments thereof and immunoglobulinchains can be recovered from the culture medium using standard proteinpurification methods. Further, expression of antibodies andantigen-binding fragments thereof and immunoglobulin chains of theinvention (or other moieties therefrom) from production cell lines canbe enhanced using a number of known techniques. For example, theglutamine synthetase gene expression system (the GS system) is a commonapproach for enhancing expression under certain conditions. The GSsystem is discussed in whole or part in connection with European PatentNos. 0 216 846, 0 256 055, and 0 323 997 and European Patent ApplicationNo. 89303964.4. Thus, in an embodiment of the invention, the mammalianhost cells (e.g., CHO) lack a glutamine synthetase gene and are grown inthe absence of glutamine in the medium wherein, however, thepolynucleotide encoding the immunoglobulin chain comprises a glutaminesynthetase gene which complements the lack of the gene in the host cell.

The present invention includes methods for purifying an anti-hRSVantibody or antigen-binding fragment thereof of the present inventioncomprising introducing a sample comprising the antibody or fragment to apurification medium (e.g., cation exchange medium, anion exchangemedium, hydrophobic exchange medium, affinity purification medium (e.g.,protein-A, protein-G, protein-A/G, protein-L)) and either collectingpurified antibody or fragment from the flow-through fraction of saidsample that does not bind to the medium; or, discarding the flow-throughfraction and eluting bound antibody or fragment from the medium andcollecting the eluate. In an embodiment of the invention, the medium isin a column to which the sample is applied. In an embodiment of theinvention, the purification method is conducted following recombinantexpression of the antibody or fragment in a host cell, e.g., wherein thehost cell is first lysed and, optionally, the lysate is purified ofinsoluble materials prior to purification on a medium.

In general, glycoproteins produced in a particular cell line ortransgenic animal will have a glycosylation pattern that ischaracteristic for glycoproteins produced in the cell line or transgenicanimal. Therefore, the particular glycosylation pattern of an antibodywill depend on the particular cell line or transgenic animal used toproduce the antibody. However, all antibodies encoded by the nucleicacid molecules provided herein, or comprising the amino acid sequencesprovided herein, comprise the instant invention, independent of theglycosylation pattern the antibodies may have. Similarly, in particularembodiments, antibodies with a glycosylation pattern comprising onlynon-fucosylated N-glycans may be advantageous, because these antibodieshave been shown to typically exhibit more potent efficacy than theirfucosylated counterparts both in vitro and in vivo (See for example,Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Pat. Nos.6,946,292 and 7,214,775). These antibodies with non-fucosylatedN-glycans are not likely to be immunogenic because their carbohydratestructures are a normal component of the population that exists in humanserum IgG.

The present invention includes bispecific and bifunctional antibodiesand antigen-binding fragments having a binding specificity for hRSV Fprotein and another antigen such as, for example, hRSV G protein, andmethods of use thereof A bispecific or bifunctional antibody is anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites. Bispecific antibodies can be producedby a variety of methods including fusion of hybridomas or linking ofFab′ fragments. See, e.g., Songsivilai, et al., (1990) Clin. Exp.Immunol. 79: 315-321, Kostelny, et al., (1992) J Immunol. 148:1547-1553.In addition, bispecific antibodies may be formed as “diabodies”(Holliger, et al., (1993) PNAS USA 90:6444-6448) or as “Janusins”(Traunecker, et al., (1991) EMBO J. 10:3655-3659 and Traunecker, et al.,(1992) Int. J. Cancer Suppl. 7:51-52).

The present invention further includes anti-hRSV F-proteinantigen-binding fragments of the anti-hRSV antibodies disclosed herein.The antibody fragments include F(ab)₂ fragments, which may be producedby enzymatic cleavage of an IgG by, for example, pepsin. Fab fragmentsmay be produced by, for example, reduction of F(ab)₂ with dithiothreitolor mercaptoethylamine.

Immunoglobulins may be assigned to different classes depending on theamino acid sequences of the constant domain of their heavy chains. Thereare at least five major classes of immunoglobulins: IgA, IgD, IgE, IgGand IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2. The inventioncomprises antibodies and antigen-binding fragments of any of theseclasses or subclasses of antibodies.

In one embodiment, the antibody or antigen-binding fragment comprises aheavy chain constant region, e.g. a human constant region, such as γ1,γ2, γ3, or γ4 human heavy chain constant region or a variant thereof. Inanother embodiment, the antibody or antigen-binding fragment comprises alight chain constant region, e.g. a human light chain constant region,such as lambda or kappa human light chain region or variant thereof. Byway of example, and not limitation the human heavy chain constant regioncan be γ4 and the human light chain constant region can be kappa. In analternative embodiment, the Fc region of the antibody is γ4 with aSer228Pro mutation (Schuurman, J et. al., 2001, Mol. Immunol. 38: 1-8).

In one embodiment, the antibody or antigen-binding fragment comprises aheavy chain constant region of the IgG1 subtype.

In some embodiments, different constant domains may be appended to V_(L)and V_(H) regions derived from the CDRs provided herein. For example, ifa particular intended use of an antibody (or fragment) of the presentinvention were to call for altered effector functions, a heavy chainconstant domain other than human IgG1 may be used, or hybrid IgG1/IgG4may be utilized.

Although human IgG1 antibodies provide for long half-life and foreffector functions, such as complement activation and antibody-dependentcellular cytotoxicity, such activities may not be desirable for all usesof the antibody. In such instances a human IgG4 constant domain, forexample, may be used. The present invention includes anti-hRSV F-proteinantibodies and antigen-binding fragments thereof which comprise an IgG4constant domain, e.g., antagonist, humanized anti-hRSV F-proteinantibodies and fragments, and methods of use thereof. In one embodiment,the IgG4 constant domain can differ from the native human IgG4 constantdomain (Swiss-Prot Accession No. P01861.1) at a position correspondingto position 228 in the EU system and position 241 in the KABAT system,where the native Ser108 is replaced with Pro, in order to prevent apotential inter-chain disulfide bond between Cys106 and Cys109(corresponding to positions Cys 226 and Cys 229 in the EU system andpositions Cys 239 and Cys 242 in the KABAT system) that could interferewith proper intra-chain disulfide bond formation. See Angal et al.(1993) Mol. Imunol. 30:105. In other instances, a modified IgG1 constantdomain which has been modified to increase half-life or reduce effectorfunction can be used.

Antibody Engineering

The antibodies of the invention may be subject to framework mutations toimprove the properties of the antibody. One such framework modificationinvolves mutating one or more residues within the framework region, oreven within one or more CDR regions, to remove T cell epitopes tothereby reduce the potential immunogenicity of the antibody. Thisapproach is also referred to as “deimmunization” and is described infurther detail in U.S. Pat. No. 7,125,689.

In particular embodiments, it will be desirable to change certain aminoacids containing exposed side-chains to another amino acid residue inorder to provide for greater chemical stability of the final antibody,so as to avoid deamidation or isomerization. The deamidation ofasparagine may occur on NG, DG, NG, NS, NA, NT, QG or QS sequences andresult in the creation of an isoaspartic acid residue that introduces akink into the polypeptide chain and decreases its stability (isoasparticacid effect). Isomerization can occur at DG, DS, DA or DT sequences. Incertain embodiments, the antibodies of the present disclosure do notcontain deamidation or asparagine isomerism sites.

For example, an asparagine (Asn) residue may be changed to Gln or Ala toreduce the potential for formation of isoaspartate at any Asn-Glysequences, particularly within a CDR. A similar problem may occur at aAsp-Gly sequence. Reissner and Aswad (2003) Cell. Mol. Life Sci.60:1281. Isoaspartate formation may debilitate or completely abrogatebinding of an antibody to its target antigen. See, Presta (2005) J.Allergy Clin. Immunol. 116:731 at 734. In one embodiment, the asparagineis changed to glutamine (Gin). It may also be desirable to alter anamino acid adjacent to an asparagine (Asn) or glutamine (Gin) residue toreduce the likelihood of deamidation, which occurs at greater rates whensmall amino acids occur adjacent to asparagine or glutamine. See,Bischoff & Kolbe (1994) J. Chromatog. 662:261. In addition, anymethionine residues (typically solvent exposed Met) in CDRs may bechanged to Lys, Leu, Ala, or Phe or other amino acids in order to reducethe possibility that the methionine sulfur would oxidize, which couldreduce antigen-binding affinity and also contribute to molecularheterogeneity in the final antibody preparation. Id. Additionally, inorder to prevent or minimize potential scissile Asn-Pro peptide bonds,it may be desirable to alter any Asn-Pro combinations found in a CDR toGln-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions aresubsequently screened to ensure that the substitutions do not decreasethe affinity or specificity of the antibody for hRSV F-protein, or otherdesired biological activity to unacceptable levels.

TABLE 2 Exemplary stabilizing CDR variants CDR Residue StabilizingVariant Sequence Asn-Gly Gln-Gly, Ala-Gly, or Asn-Ala (N-G) (Q-G),(A-G), or (N-A) Asp-Gly Glu-Gly, Ala-Gly or Asp-Ala (D-G) (E-G), (A-G),or (D-A) Met (typically solvent Lys, Leu, Ala, or Phe exposed) (M) (K),(L), (A), or (F) Asn Gln or Ala (N) (Q) or (A) Asn-Pro Gln-Pro, Ala-Pro,or Asn-Ala (N-P) (Q-P), (A-P), or (N-A)

Antibody Engineering of the Fc Region

The antibodies and antigen-binding fragments thereof disclosed herein(e.g., RB1) can also be engineered to include modifications within theFc region, typically to alter one or more properties of the antibody,such as serum half-life, complement fixation, Fc receptor binding,and/or effector function (e.g., antigen-dependent cellularcytotoxicity). Furthermore, the antibodies and antigen-binding fragmentsthereof disclosed herein (e.g., RB1) can be chemically modified (e.g.,one or more chemical moieties can be attached to the antibody) or bemodified to alter its glycosylation, again to alter one or moreproperties of the antibody or fragment. Each of these embodiments isdescribed in further detail below. The numbering of residues in the Fcregion is that of the EU index of Kabat.

The antibodies and antigen-binding fragments thereof disclosed herein(e.g., RB1) also include antibodies and fragments with modified (orblocked) Fc regions to provide altered effector functions. See, e.g.,U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571; WO2006/0057702.Such modifications can be used to enhance or suppress various reactionsof the immune system, with possible beneficial effects in diagnosis andtherapy. Alterations of the Fc region include amino acid changes(substitutions, deletions and insertions), glycosylation ordeglycosylation, and adding multiple Fc regions. Changes to the Fc canalso alter the half-life of antibodies in therapeutic antibodies,enabling less frequent dosing and thus increased convenience anddecreased use of material. See Presta, 2005, J. Allergy Clin. Immunol.116:731 at 734-35.

In one embodiment of the invention, the hinge region of CH1 is modifiedsuch that the number of cysteine residues in the hinge region isincreased or decreased. This approach is described further in U.S. Pat.No. 5,677,425. The number of cysteine residues in the hinge region ofCH1 is altered, for example, to facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the antibody or antigen-binding fragment of theinvention (e.g., RB1) is modified to increase its biological half-life.Various approaches are possible. For example, one or more of thefollowing mutations can be introduced: T252L, T254S, T256F, as describedin U.S. Pat. No. 6,277,375. Alternatively, to increase the biologicalhalf-life, the antibody can be altered within the CH1 or CL region tocontain a salvage receptor binding epitope taken from two loops of a CH2domain of an Fc region of an IgG, as described in U.S. Pat. Nos.5,869,046 and 6,121,022. In one embodiment, a M252Y/S254T/T256E (YTE)mutation is introduced. See, e.g., Oganesyan et al., Mol. Immunol. 2009,46:1750.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody or antigen-binding fragment. Forexample, one or more amino acids selected from amino acid residues 234,235, 236, 237, 297, 318, 320 and 322 can be replaced with a differentamino acid residue such that the antibody has an altered affinity for aneffector ligand and retains the antigen-binding ability of the parentantibody. The effector ligand to which affinity is altered can be, forexample, an Fc receptor or the Cl component of complement. This approachis described in further detail in U.S. Pat. Nos. 5,624,821 and5,648,260.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further inInternational Patent Application Publication No. WO 94/29351.

In yet another example, the Fc region is modified to decrease theability of the antibody or antigen-binding fragment of the invention(e.g., RB1) to mediate antibody dependent cellular cytotoxicity (ADCC)and/or to decrease the affinity of the antibody or fragment for an Fcyreceptor by modifying one or more amino acids at the followingpositions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265,267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292,293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322,324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373,376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or439. This approach is described further in International PatentApplication Publication No. WO 00/42072. Moreover, the binding sites onhuman IgG1 for FcγR1, FcγRII, FcγRIII and FcRn have been mapped andvariants with improved binding have been described (see Shields et al.(2001) J. Biol. Chem. 276:6591-6604).

In one embodiment of the invention, the Fc region is modified todecrease the ability of the antibody of the invention (e.g., RB1) tomediate effector function and/or to increase anti-inflammatoryproperties by modifying residues 243 and 264. In one embodiment, the Fcregion of the antibody or fragment is modified by changing the residuesat positions 243 and 264 to alanine. In one embodiment, the Fc region ismodified to decrease the ability of the antibody or fragment to mediateeffector function and/or to increase anti-inflammatory properties bymodifying residues 243, 264, 267 and 328.

Effector Function Enhancement

In some embodiments, the Fc region of an anti-hRSV antibody is modifiedto increase the ability of the antibody or antigen-binding fragment tomediate effector function and/or to increase their binding to theFcgamma receptors (FcγRs).

The term “Effector Function” as used herein is meant to refer to one ormore of Antibody Dependent Cell mediated Cytotoxic activity (ADCC),Complement-dependent cytotoxic activity (CDC) mediated responses,Fc-mediated phagocytosis or antibody dependent cellular phagocytosis(ADCP) and antibody recycling via the FcRn receptor.

The interaction between the constant region of an antigen bindingprotein and various Fc receptors (FcR) including FcgammaRI (CD64),FcgammaRII (CD32) and FcgammaRIII (CD16) is believed to mediate theeffector functions, such as ADCC and CDC, of the antigen bindingprotein. The Fc receptor is also important for antibody cross-linking,which can be important for anti-tumor immunity.

Effector function can be measured in a number of ways including forexample via binding of the FcγRIII to Natural Killer cells or via FcγRIto monocytes/macrophages to measure for ADCC effector function. Forexample an antigen binding protein of the present invention can beassessed for ADCC effector function in a Natural Killer cell assay.Examples of such assays can be found in Shields et al., 2001 J. Biol.Chem., Vol. 276, p 6591-6604; Chappel et al., 1993 J. Biol. Chem. 268:25124-25131; Lazar et al., 2006, Proc Natl Acad Sci USA 103:4005-4010.

The ADCC or CDC properties of antibodies of the present invention, ortheir cross-linking properties, may be enhanced in a number of ways.

Human IgG1 constant regions containing specific mutations or alteredglycosylation on residue Asn297 have been shown to enhance binding to Fcreceptors. In some cases these mutations have also been shown to enhanceADCC and CDC (Lazar et al., Proc Natl Acad Sci USA 2006, 103:4005-4010;Shields et al., J Biol Chem 2001, 276:6591-6604; Nechansky et al., MolImmunol 2007, 44:1815-1817).

In one embodiment of the present invention, such mutations are in one ormore of positions selected from 239, 332 and 330 (IgG1), or theequivalent positions in other IgG isotypes. Examples of suitablemutations are S239D and I332E and A330L. In one embodiment, the antigenbinding protein of the invention herein described is mutated atpositions 239 and 332, for example S239D and I332E or in a furtherembodiment it is mutated at three or more positions selected from 239and 332 and 330, for example S239D and I332E and A330L. (EU indexnumbering).

In an alternative embodiment of the present invention, there is providedan antibody comprising a heavy chain constant region with an alteredglycosylation profile such that the antigen binding protein has enhancedeffector function. For example, wherein the antibody has enhanced ADCCor enhanced CDC or wherein it has both enhanced ADCC and CDC effectorfunction. Examples of suitable methodologies to produce antigen bindingproteins with an altered glycosylation profile are described inInternational Patent Application Publication Nos. WO2003011878 andWO2006014679 and European Patent Application No. EP1229125.

In a further aspect, the present invention provides “non-fucosylated” or“afucosylated” antibodies. Non-fucosylated antibodies harbor atri-mannosyl core structure of complex-type N-glycans of Fc withoutfucose residue. These glycoengineered antibodies that lack core fucoseresidue from the Fc N-glycans may exhibit stronger ADCC than fucosylatedequivalents due to enhancement of FcγRIIIa binding capacity.

The present invention also provides a method for the production of anantibody according to the invention comprising the steps of: a)culturing a recombinant host cell comprising an expression vectorcomprising an isolated nucleic acid as described herein, wherein therecombinant host cell does not comprise an alpha-1,6-fucosyltransferase;and b) recovering the antigen binding protein. The recombinant host cellmay be not normally contain a gene encoding analpha-1,6-fucosyltransferase (for example yeast host cells such asPichia sp.) or may have been genetically modified to inactive analpha-1,6-fucosyltransferase. Recombinant host cells which have beengenetically modified to inactivate the FUT8 gene encoding analpha-1,6-fucosyltransferase are available. See, e.g., the POTELLIGENT™technology system available from BioWa, Inc. (Princeton, N.J.) in whichCHOK1SV cells lacking a functional copy of the FUT8 gene producemonoclonal antibodies having enhanced antibody dependent cell mediatedcytotoxicity (ADCC) activity that is increased relative to an identicalmonoclonal antibody produced in a cell with a functional FUT8 gene.Aspects of the POTELLIGENT™ technology system are described in U.S. Pat.Nos. 7,214,775; 6,946,292; and International Patent Application Nos.WO0061739 and WO0231240. Those of ordinary skill in the art will alsorecognize other appropriate systems.

It will be apparent to those skilled in the art that such modificationsmay not only be used alone but may be used in combination with eachother in order to further enhance effector function.

Production of Antibodies with Modified Glycosylation

In still another embodiment, the antibodies or antigen-binding fragmentsof the invention (e.g., RB1) comprise a particular glycosylationpattern. For example, an afucosylated or an aglycosylated antibody orfragment can be made (i.e., the antibody lacks fucose or glycosylation,respectively). The glycosylation pattern of an antibody or fragment maybe altered to, for example, increase the affinity or avidity of theantibody or fragment for a hRSV F-protein antigen. Such modificationscan be accomplished by, for example, altering one or more of theglycosylation sites within the antibody or fragment sequence. Forexample, one or more amino acid substitutions can be made that resultremoval of one or more of the variable region framework glycosylationsites to thereby eliminate glycosylation at that site. Suchaglycosylation may increase the affinity or avidity of the antibody orfragment for antigen. See, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861.

Antibodies and antigen-binding fragments disclosed herein (e.g., RB1)may further include those produced in lower eukaryote host cells, inparticular fungal host cells such as yeast and filamentous fungi havebeen genetically engineered to produce glycoproteins that havemammalian- or human-like glycosylation patterns (See for example, Choiet al, (2003) Proc. Natl. Acad. Sci. USA 100: 5022-5027; Hamilton etal., (2003) Science 301: 1244-1246; Hamilton et al., (2006) Science 313:1441-1443; Nett et al., (2011) Yeast 28(3):237-52; Hamilton et al.,(2007) Curr Opin Biotechnol. October; 18(5):387-92). These geneticallymodified host cells have the ability to control the glycosylationprofile of glycoproteins that are produced in the cells such thatcompositions of glycoproteins can be produced wherein a particularN-glycan structure predominates (see, e.g., U.S. Pat. Nos. 7,029,872 and7,449,308). These genetically modified host cells have been used toproduce antibodies that have predominantly particular N-glycanstructures (See for example, Li et al., (2006) Nat. Biotechnol. 24:210-215).

In particular embodiments, the antibodies and antigen-binding fragmentsthereof disclosed herein (e.g., RB1) further include those produced inlower eukaryotic host cells and which comprise fucosylated andnon-fucosylated hybrid and complex N-glycans, including bisected andmultiantennary species, including but not limited to N-glycans such asGlcNAc₍₁₋₄₎Man₃GlcNAc₂; Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂;NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂.

In particular embodiments, the antibodies and antigen-binding fragmentsthereof provided herein (e.g., RB1) may comprise antibodies or fragmentshaving at least one hybrid N-glycan selected from the group consistingof GlcNAcMan₅GlcNAc₂; GalGlcNAcMan₅GlcNAc₂; andNANAGalGlcNAcMan₅GlcNAc₂. In particular aspects, the hybrid N-glycan isthe predominant N-glycan species in the composition.

In particular embodiments, the antibodies and antigen-binding fragmentsthereof provided herein (e.g., RB1) comprise antibodies and fragmentshaving at least one complex N-glycan selected from the group consistingof GlcNAcMan₃GlcNAc₂; GalGlcNAcMan₃GlcNAc₂; NANAGalGlcNAcMan₃GlcNAc₂;GlcNAc₂Man₃GlcNAc₂; GalGlcNAc₂Man₃GlcNAc₂; Gal₂GlcNAc₂Man₃GlcNAc₂;NANAGal₂GlcNAc₂Man₃GlcNAc₂; and NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. Inparticular aspects, the complex N-glycan are the predominant N-glycanspecies in the composition. In further aspects, the complex N-glycan isa particular N-glycan species that comprises about 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycans inthe composition. In one embodiment, the antibody and antigen bindingfragments thereof provided herein comprise complex N-glycans, wherein atleast 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of thecomplex N-glycans in comprise the structure NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂,wherein such structure is afucosylated. Such structures can be produced,e.g., in engineered Pichia pastoris host cells.

In particular embodiments, the N-glycan is fucosylated. In general, thefucose is in an α1,3-linkage with the GlcNAc at the reducing end of theN-glycan, an α1,6-linkage with the GlcNAc at the reducing end of theN-glycan, an a1,2-linkage with the Gal at the non-reducing end of theN-glycan, an α1,3-linkage with the GlcNac at the non-reducing end of theN-glycan, or an α1,4-linkage with a GlcNAc at the non-reducing end ofthe N-glycan.

Therefore, in particular aspects of the above the glycoproteincompositions, the glycoform is in an α1,3-linkage or α1,6-linkage fucoseto produce a glycoform selected from the group consisting ofMan₅GlcNAc₂(Fuc), GlcNAcMan₅GlcNAc₂(Fuc), Man₃GlcNAc₂(Fuc),GlcNAcMan₃GlcNAc₂(Fuc), GlcNAc₂Man₃GlcNAc₂(Fuc),GalGlcNAc₂Man₃GlcNAc₂(Fuc), Gal₂GlcNAc₂Man₃GlcNAc₂(Fuc),NANAGal₂GlcNAc₂Man₃GlcNAc₂(Fuc), and NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂(Fuc);in an α1,3-linkage or α1,4-linkage fucose to produce a glycoformselected from the group consisting of GlcNAc(Fuc)Man₅GlcNAc₂,GlcNAc(Fuc)Man₃GlcNAc₂, GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂,GalGlcNAc₂(Fuc₁₋₂₎Man₃GlcNAc₂, Gal₂GlcNAc₂(Fuc1-2)Man3GlcNAc2,NANAGal2GlcNAc2(Fuc₁₋₂₎Man₃GlcNAc₂, andNANA₂Gal₂GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂; or in an α1,2-linkage fucose toproduce a glycoform selected from the group consisting ofGal(Fuc)GlcNAc₂Man₃GlcNAc₂, Gal₂(Fuc₁₋₂₎GlcNAc₂Man₃GlcNAc₂,NANAGal₂(Fuc₁₋₂₎GlcNAc₂Man₃GlcNAc₂, andNANA₂Gal₂(Fuc₁₋₂₎GlcNAc₂Man₃GlcNAc₂.

In further aspects, the antibodies or antigen-binding fragments thereofcomprise high mannose N-glycans, including, but not limited to,Man₈GlcNAc₂, Man₇GlcNAc₂, Man₆GlcNAc₂, Man₅GlcNAc₂, Man₄GlcNAc₂, orN-glycans that consist of the Man₃GlcNAc₂ N-glycan structure.

In further aspects of the above, the complex N-glycans further includefucosylated and non-fucosylated bisected and multiantennary species.

As used herein, the terms “N-glycan” and “glycoform” are usedinterchangeably and refer to an N-linked oligosaccharide, for example,one that is attached by an asparagine-N-acetylglucosamine linkage to anasparagine residue of a polypeptide. N-linked glycoproteins contain anN-acetylglucosamine residue linked to the amide nitrogen of anasparagine residue in the protein. The predominant sugars found onglycoproteins are glucose, galactose, mannose, fucose,N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialicacid (e.g., N-acetyl-neuraminic acid (NANA)). The processing of thesugar groups occurs co-translationally in the lumen of the ER andcontinues post-translationally in the Golgi apparatus for N-linkedglycoproteins.

N-glycans have a common pentasaccharide core of Man₃GlcNAc₂ (“Man”refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). Usually, N-glycanstructures are presented with the non-reducing end to the left and thereducing end to the right. The reducing end of the N-glycan is the endthat is attached to the Asn residue comprising the glycosylation site onthe protein. N-glycans differ with respect to the number of branches(antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucoseand sialic acid) that are added to the Man₃GlcNAc₂ (“Man3”) corestructure which is also referred to as the “trimannose core”, the“pentasaccharide core” or the “paucimannose core”. N-glycans areclassified according to their branched constituents (e.g., high mannose,complex or hybrid). A “high mannose” type N-glycan has five or moremannose residues. A “complex” type N-glycan typically has at least oneGlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attachedto the 1,6 mannose arm of a “trimannose” core. Complex N-glycans mayalso have galactose (“Gal”) or N-acetylgalactosamine (“GalNAc”) residuesthat are optionally modified with sialic acid or derivatives (e.g.,“NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refersto acetyl). Complex N-glycans may also have intrachain substitutionscomprising “bisecting” GlcNAc and core fucose (“Fuc”). Complex N-glycansmay also have multiple antennae on the “trimannose core,” often referredto as “multiple antennary glycans.” A “hybrid” N-glycan has at least oneGlcNAc on the terminal of the 1,3 mannose arm of the trimannose core andzero or more mannoses on the 1,6 mannose arm of the trimannose core. Thevarious N-glycans are also referred to as “glycoforms.”

With respect to complex N-glycans, the terms “G-2”, “G-1”, “G0”, “G1”,“G2”, “A1”, and “A2” mean the following. “G-2” refers to an N-glycanstructure that can be characterized as Man₃GlcNAc₂; the term “G-1”refers to an N-glycan structure that can be characterized asGlcNAcMan₃GlcNAc₂; the term “G0” refers to an N-glycan structure thatcan be characterized as GlcNAc₂Man₃GlcNAc₂; the term “G1” refers to anN-glycan structure that can be characterized as GalGlcNAc₂Man₃GlcNAc₂;the term “G2” refers to an N-glycan structure that can be characterizedas Gal₂GlcNAc₂Man₃GlcNAc₂; the term “A1” refers to an N-glycan structurethat can be characterized as NANAGal₂GlcNAc₂Man₃GlcNAc₂; and, the term“A2” refers to an N-glycan structure that can be characterized asNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. Unless otherwise indicated, the terms“G-2”, “G-1”, “G0”, “G1”, “G2”, “A1”, and “A2” refer to N-glycan speciesthat lack fucose attached to the GlcNAc residue at the reducing end ofthe N-glycan. When the term includes an “F”, the “F” indicates that theN-glycan species contains a fucose residue on the GlcNAc residue at thereducing end of the N-glycan. For example, G0F, G1F, G2F, A1F, and A2Fall indicate that the N-glycan further includes a fucose residueattached to the GlcNAc residue at the reducing end of the N-glycan.Lower eukaryotes such as yeast and filamentous fungi do not normallyproduce N-glycans that produce fucose.

With respect to multiantennary N-glycans, the term “multiantennaryN-glycan” refers to N-glycans that further comprise a GlcNAc residue onthe mannose residue comprising the non-reducing end of the 1,6 arm orthe 1,3 arm of the N-glycan or a GlcNAc residue on each of the mannoseresidues comprising the non-reducing end of the 1,6 arm and the 1,3 armof the N-glycan. Thus, multiantennary N-glycans can be characterized bythe formulas GlcNAc₍₂₋₄₎Man₃GlcNAc₂, Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂, orNANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. The term “1-4” refers to 1, 2,3, or 4 residues.

With respect to bisected N-glycans, the term “bisected N-glycan” refersto N-glycans in which a GlcNAc residue is linked to the mannose residueat the reducing end of the N-glycan. A bisected N-glycan can becharacterized by the formula GlcNAc₃Man₃GlcNAc₂ wherein each mannoseresidue is linked at its non-reducing end to a GlcNAc residue. Incontrast, when a multiantennary N-glycan is characterized asGlcNAc₃Man₃GlcNAc₂, the formula indicates that two GlcNAc residues arelinked to the mannose residue at the non-reducing end of one of the twoarms of the N-glycans and one GlcNAc residue is linked to the mannoseresidue at the non-reducing end of the other arm of the N-glycan.

Antibody Physical Properties

The antibodies and antigen-binding fragments thereof disclosed herein(e.g., RB1) may further contain one or more glycosylation sites ineither the light or heavy chain immunoglobulin variable region. Certainglycosylation sites can result in decreased immunogenicity of theantibody or fragment or an alteration of the pK of the antibody due toaltered antigen-binding (Marshall et al., 1972, Annu Rev Biochem41:673-702; Gala and Morrison, 2004, J Immunol 172:5489-94; Wallick etal., 1988, J Exp Med 168:1099-109; Spiro, 2002, Glycobiology 12:43R-56R;Parekh et al., 1985, Nature 316:452-7; Mimura et al., 2000, Mol Immunol37:697-706). Glycosylation has been known to occur at motifs containingan N-X-S/T sequence.

Each antibody or antigen-binding fragment (e.g., RB1) will have a uniqueisoelectric point (pI), which generally falls in the pH range between 6and 9.5. The pI for an IgG1 antibody typically falls within the pH rangeof 7-9.5 and the pI for an IgG4 antibody typically falls within the pHrange of 6-8.

Each antibody or antigen-binding fragment (e.g., RB1) will have acharacteristic melting temperature, with a higher melting temperatureindicating greater overall stability in vivo (Krishnamurthy R andManning MC (2002) Curr Pharm Biotechnol 3:361-71). In general, theT_(M1) (the temperature of initial unfolding) may be greater than 60°C., greater than 65° C., or greater than 70° C. The melting point of anantibody or fragment can be measured using differential scanningcalorimetry (Chen et al., (2003) Pharm Res 20:1952-60; Ghirlando et al.,(1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al.,(2002) J. Chromatogr Sci 40:343-9).

In a further embodiment, antibodies and antigen-binding fragmentsthereof (e.g., RB1) are selected that do not degrade rapidly.Degradation of an antibody or fragment can be measured using capillaryelectrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995)Anal Chem 67:3626-32).

In a further embodiment, antibodies (e.g., RB1) and antigen-bindingfragments thereof are selected that have minimal aggregation effects,which can lead to the triggering of an unwanted immune response and/oraltered or unfavorable pharmacokinetic properties. Generally, antibodiesand fragments are acceptable with aggregation of 25% or less, 20% orless, 15% or less, 10% or less, or 5% or less. Aggregation can bemeasured by several techniques, including size-exclusion column (SEC),high performance liquid chromatography (HPLC), and light scattering.

Antibody Conjugates

The anti-hRSV F-protein antibodies and antigen-binding fragments thereofdisclosed herein (e.g., RB1) may also be conjugated to a chemicalmoiety. The chemical moiety may be, inter alia, a polymer, aradionuclide or a cytotoxic factor. In particular embodiments, thechemical moiety is a polymer which increases the half-life of theantibody or fragment in the body of a subject. Suitable polymersinclude, but are not limited to, hydrophilic polymers which include butare not limited to polyethylene glycol (PEG) (e.g., PEG with a molecularweight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa),dextran and monomethoxypolyethylene glycol (mPEG). Lee, et al., (1999)(Bioconj. Chem. 10:973-981) discloses PEG conjugated single-chainantibodies. Wen, et al., (2001) (Bioconj. Chem. 12:545-553) discloseconjugating antibodies with PEG which is attached to a radiometalchelator (diethylenetriaminpentaacetic acid (DTPA)).

The antibodies and antigen-binding fragments thereof disclosed herein(e.g., RB1) may also be conjugated with labels such as ⁹⁹Tc, ⁹⁰Y, ¹¹¹In,³²P, ¹⁴C, ¹²⁵I, ³H, ¹³¹I, ¹¹C, ¹⁵O, ¹³N, ¹⁸F, ³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra,⁶⁰Co, ⁵⁹Fe, ⁵⁷Se, ¹⁵²Eu, ⁶⁷CU, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th,and ⁴⁰K, ¹⁵⁷Gd, ⁵⁵Mn, ⁵²Tr, and ⁵⁶Fe.

The antibodies and antigen-binding fragments disclosed herein (e.g.,RB1) may also be PEGylated, for example to increase its biological(e.g., serum) half-life. To PEGylate an antibody or fragment, theantibody or fragment, typically is reacted with a reactive form ofpolyethylene glycol (PEG), such as a reactive ester or aldehydederivative of PEG, under conditions in which one or more PEG groupsbecome attached to the antibody or antibody fragment. In particularembodiments, the PEGylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C₁-C₁₀) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody or fragment to be PEGylated is anaglycosylated antibody or fragment. Methods for PEGylating proteins areknown in the art and can be applied to the antibodies of the invention.See, e.g., European Patent Application Nos. EP 0 154 316 and EP 0 401384.

The antibodies and antigen-binding fragments disclosed herein (e.g.,RB1) may also be conjugated with fluorescent or chemilluminescentlabels, including fluorophores such as rare earth chelates, fluoresceinand its derivatives, rhodamine and its derivatives, isothiocyanate,phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde,fluorescamine, ¹⁵²Eu, dansyl, umbelliferone, luciferin, luminal label,isoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridimium salt label, an oxalate ester label, an aequorinlabel, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels andstable free radicals.

The antibodies and antigen-binding fragments thereof of the invention(e.g., RB1) may also be conjugated to a cytotoxic factor such asdiptheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteinsand compounds (e.g., fatty acids), dianthin proteins, Phytoiaccaamericana proteins PAPI, PAPII, and PAP-S, Momordica charantiainhibitor, curcin, crotin, Saponaria officinalis inhibitor, mitogellin,restrictocin, phenomycin, and enomycin.

Any method known in the art for conjugating the antibodies andantigen-binding fragments thereof of the invention (e.g., RB1) to thevarious moieties may be employed, including those methods described byHunter et al., 1962, Nature 144:945; David et al., 1974, Biochemistry13:1014; Pain et al., 1981, J. Immunol. Meth. 40:219; and Nygren, 1982,Histochem. and Cytochem. 30:407. Methods for conjugating antibodies andfragments are conventional and very well known in the art.

Prophylactic and Therapeutic Uses of Anti-hRSV Antibodies

Further provided are methods for preventing, treating or ameliorating asymptom of RSV infection in subjects, including human subjects, in needof such prevention, treatment, or amelioration with the isolatedantibodies or antigen-binding fragments thereof disclosed herein (e.g.,RB1). In one embodiment of the invention, such subject suffers from aRSV infection. In one embodiment of the invention, such subject is atrisk of a RSV infection.

In a specific embodiment, a mammal, preferably a human, is administereda prophylactic, therapeutic or pharmaceutical composition comprising oneor more antibodies of the present invention or fragments thereof for thetreatment, prevention or amelioration of one or more symptoms associatedwith a RSV infection in an amount effective for decreasing RSV titers.In accordance with this embodiment, an effective amount of antibodies orantibody fragments reduces the RSV titers in the lung as measured, forexample, by the concentration of RSV in sputum samples or a lavage fromthe lungs from a mammal. In another embodiment, a mammal, preferably ahuman, is administered a prophylactic, therapeutic or pharmaceuticalcomposition comprising one or more antibodies of the present inventionor fragments thereof for the treatment, prevention or amelioration ofsymptoms associated with a RSV infection in an amount effective forneutralizing RSV and/or blocking RSV infection in the mammal.

The monoclonal antibodies or antigen binding fragments thereof can alsobe used immunotherapeutically for RSV disease in both humans and otheranimals. The term, “immunotherapeutically” or “immunotherapy” as usedherein in conjunction with the monoclonal antibodies or antigen bindingfragments thereof of the invention denotes both prophylactic as well astherapeutic administration and both passive immunization withsubstantially purified polypeptide products, as well as gene therapy bytransfer of polynucleotide sequences encoding the product or partthereof. Passive immunization includes transfer of active humoralimmunity or providing antibodies to a subject in need thereof.Accordingly, in certain embodiments of the invention, the presentinvention provides methods for transfer of active humoral immunity andmethods of providing RSV antibodies or antigen binding fragmentsthereof, such as IgG antibodies, to a patient at risk of RSV infection.Thus, the monoclonal antibodies or antigen binding fragments thereof canbe administered to high-risk subjects in order to lessen the likelihoodand/or severity of RSV disease or administered to subjects alreadyevidencing active RSV infection.

The present invention also provides a method for modulating or treatingat least one adult or pediatric RSV related disease, in a cell, tissue,organ, animal, or patient including, but not limited to, lowerrespiratory infections, pneumonia, tracheobronchitis, bronchiolitis,bronchitis, and any related infections or inflammatory disorders, suchas but not limited to at least one of, or at least one inflammationrelated to, systemic inflammatory response syndrome, sepsis syndrome,gram positive sepsis, gram negative sepsis, culture negative sepsis,fungal sepsis, neutropenic fever, urosepsis, meningococcemia, adultrespiratory distress syndrome, allergic rhinitis, perennial rhinitis,asthma, systemic anaphalaxis, receptor hypersensitivity reactions,chronic obstructive pulmonary disease (COPD), hypersensitivitypneumonitis, granulomas due to intracellular organisms, drugsensitivity, cachexia, cystic fibrosis, neonatal chronic lung disease;at least one infectious disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: acute orchronic bacterial infection, acute and chronic parasitic or infectiousprocesses, including bacterial, viral and fungal infections, HIVinfection, HIV neuropathy, meningitis, hepatitis (A,B or C, or thelike), septic arthritis, peritonitis, pneumonia, epiglottitis, E. coli0157:h7, hemolytic uremic syndrome, thrombolytic thrombocytopenicpurpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,mycobacterium tuberculosis, mycobacterium avium intracellulare,pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis,epidydimitis, legionella, lyme disease, influenza A, Epstein-Barr virus,vital-associated hemaphagocytic syndrome, vital encephalitis, asepticmeningitis, and the like. Such a method can optionally compriseadministering an effective amount of a composition or pharmaceuticalcomposition comprising at least one RSV antibody or antigenic fragmentthereof to a cell, tissue, organ, animal or patient in need of suchmodulation, treatment or therapy.

In one embodiment, prophylactic, therapeutic or pharmaceuticalcompositions comprising antibodies of the invention or fragments thereofare administered to a mammal, preferably a human, to treat, prevent orameliorate one or more symptoms associated with RSV infection. Inanother embodiment, prophylactic, therapeutic or pharmaceuticalcompositions comprising antibodies of the invention or fragments thereofare administered to a human with cystic fibrosis, bronchopulmonarydysplasia, congenital heart disease, congenital immunodeficiency oracquired immunodeficiency, or to a human who has had a transplant (e.g.,bone marrow, lung, or hematopoietic stem cell transplantation (HSCT)) totreat, prevent or ameliorate one or more symptoms associated with RSVinfection.

In another embodiment, prophylactic, therapeutic or pharmaceuticalcompositions comprising antibodies of the invention or fragments thereofare administered to a human infant, preferably a human infant bornprematurely or a human infant at risk of hospitalization for RSVinfection to treat, prevent or ameliorate one or more symptomsassociated with RSV infection. In yet another embodiment, prophylactic,therapeutic or pharmaceutical compositions comprising antibodies of theinvention or fragments thereof are administered to the elderly or peoplein group homes (e.g., nursing homes or rehabilitation centers) orimmunocompromised individuals.

It is preferred to use high affinity and/or potent in vivo inhibitingantibodies and/or neutralizing antibodies or antigen binding fragmentsthereof that immunospecifically bind to a RSV antigen, for bothprevention of RSV infection and therapy for RSV infection. It is alsopreferred to use polynucleotides encoding high affinity and/or potent invivo inhibiting antibodies and/or neutralizing antibodies or antigenbinding fragments thereof that immunospecifically bind to a RSV antigen,for both prevention of RSV infection and therapy for RSV infection. Suchantibodies or fragments thereof will preferably have an affinity for theRSV F glycoprotein and/or fragments of the F glycoprotein.

Antibodies and functional equivalents (such as antigen binding fragmentsthereof) according to the present invention recognize an epitope withinRSV F protein. Antibodies or functional equivalents thereof thatspecifically recognize said epitope can be combined with RSV-specificantibodies that bind to a different epitope that are already known, suchas palivizumab, D25, AM14, AM16 and AM23. By combining an antibody orfunctional equivalent according to the invention that specificallyrecognizes said epitope with a known RSV-specific antibody, two or moredifferent epitopes of RSV are recognized during the same therapy. Thisway, a stronger immunogenic response to RSV and/or a higher antibodyspecificity against RSV can be reached. With a stronger immunogenicresponse to and higher specificity against RSV, such combination mayresult in more effective treatment and/or prevention of a RSV-relateddisorder.

One or more antibodies of the present invention or fragments thereofthat immunospecifically bind to one or more RSV antigens may be usedlocally or systemically in the body as a prophylactic. The antibodies ofthis invention or fragments thereof may also be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3,IL-7, and IL-15), which, for example, serve to increase the number oractivity of effector cells which interact with the antibodies. Theantibodies of this invention or fragments thereof may also beadvantageously utilized in combination with other monoclonal or chimericantibodies, or with lymphokines or hematopoietic growth factors (suchas, e.g., IL-2, IL-3 and IL-7), which, for example, serve to increasethe immune response. The antibodies of this invention or fragmentsthereof may also be advantageously utilized in combination with one ormore drugs used to treat RSV infection such as, for example anti-viralagents.

Antibodies of the invention or fragments may be used in combination withone or more of the following drugs: NIH-351 (Gemini Technologies),recombinant RSV vaccine (Aviron), RSVf-2 (Intracel), F-50042 (PierreFabre), T-786 (Trimeris), VP-36676 (ViroPharma), RFI-641 (American HomeProducts), VP-14637 (ViroPharma), PFP-1 and PFP-2 (American HomeProducts), RSV vaccine (Avant Immunotherapeutics), and F-50077 (PierreFabre).

The antibodies or antigen binding fragments thereof of the invention maybe administered alone or in combination with other types of treatments(e.g., hormonal therapy, immunotherapy, and anti-inflammatory agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, human orhumanized antibodies, fragments derivatives, analogs, or nucleic acids,are administered to a human patient for therapy or prophylaxis.

A “subject” may be a mammal such as a human, dog, cat, horse, cow,mouse, rat, monkey (e.g., cynomolgous monkey, e.g., Macaca fascicularis)or rabbit. In preferred embodiments of the invention, the subject is ahuman subject.

In particular embodiments, the antibodies or antigen-binding fragmentsthereof disclosed herein (e.g., RB1) may be used alone, or inassociation with antiviral therapy.

In particular embodiments, the antibodies or antigen-binding fragmentsthereof disclosed herein (e.g., RB1) may be used alone, or inassociation with another RSV monoclonal antibody.

In particular embodiments, the antibodies or antigen-binding fragmentsthereof disclosed herein (e.g., RB1) may be used alone, or inassociation with another RSV vaccine.

The term “in association with” indicates that the componentsadministered in a method of the present invention (e.g., an anti-hRSVantibody or antigen-binding fragment thereof (e.g., RB1) along with,e.g., palivizumab) can be formulated into a single composition forsimultaneous delivery or formulated separately into two or morecompositions (e.g., a kit). Each component can be administered to asubject at a different time than when the other component isadministered; for example, each administration may be givennon-simultaneously (e.g., separately or sequentially) at severalintervals over a given period of time. Moreover, the separate componentsmay be administered to a subject by the same or by a different route.

Experimental and Diagnostic Uses

The anti-hRSV F protein antibodies and antigen-binding fragments thereofdisclosed herein (e.g., RB1) may be used as affinity purificationagents. In this process, the anti-hRSV F protein antibodies andantigen-binding fragments thereof are immobilized on a solid phase sucha Sephadex®, glass or agarose resin or filter paper, using methods wellknown in the art. The immobilized antibody or fragment is contacted witha sample containing the hRSV F protein (or a fragment thereof) to bepurified, and thereafter the support is washed with a suitable solventthat will remove substantially all the material in the sample except thehRSV F protein, which is bound to the immobilized antibody or fragment.Finally, the support is washed with a solvent which elutes the boundhRSV F protein (e.g., protein A). Such immobilized antibodies andfragments form part of the present invention.

Further provided are antigens for generating secondary antibodies whichare useful for example for performing Western blots and otherimmunoassays discussed herein. In particular, polypeptides are disclosedwhich comprise the variable regions and/or CDR sequences of atherapeutic antibody disclosed herein (e.g., RB1) and which may be usedto generate anti-idiotypic antibodies for use in specifically detectingthe presence of the antibody, e.g., in a therapeutic context.

Anti-hRSV F protein antibodies and antigen-binding fragments thereof mayalso be useful in diagnostic assays for hRSV F protein, e.g., detectingits expression in specific cells, tissues, or serum. Such diagnosticmethods may be useful in various disease diagnoses.

The present invention includes ELISA assays (enzyme-linked immunosorbentassay) incorporating the use of an anti-hRSV F protein antibody orantigen-binding fragment thereof disclosed herein (e.g., RB1).

For example, such a method comprises the following steps:

(a) coat a substrate (e.g., surface of a microtiter plate well, e.g., aplastic plate) with anti-hRSV F protein antibody or antigen-bindingfragment thereof;

(b) apply a sample to be tested for the presence of RSV F protein to thesubstrate;

(c) wash the plate, so that unbound material in the sample is removed;

(d) apply detectably labeled antibodies (e.g., enzyme-linked antibodies)which are also specific to the RSV F protein antigen;

(e) wash the substrate, so that the unbound, labeled antibodies areremoved;

(f) if the labeled antibodies are enzyme linked, apply a chemical whichis converted by the enzyme into a fluorescent signal; and

(g) detect the presence of the labeled antibody.

Detection of the label associated with the substrate indicates thepresence of the hRSV F protein.

In a further embodiment, the labeled antibody or antigen-bindingfragment thereof is labeled with peroxidase which react with ABTS (e.g.,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) or3,3′,5,5′-Tetramethylbenzidine to produce a color change which isdetectable. Alternatively, the labeled antibody or fragment is labeledwith a detectable radioisotope (e.g., ³H) which can be detected byscintillation counter in the presence of a scintillant.

An anti-hRSV F protein antibody or antigen-binding fragment thereof ofthe invention (e.g., RB1) may be used in a Western blot orimmune-protein blot procedure. Such a procedure forms part of thepresent invention and includes e.g.:

(1) optionally transferring proteins from a sample to be tested for thepresence of hRSV F protein (e.g., from a PAGE or SDS-PAGEelectrophoretic separation of the proteins in the sample) onto amembrane or other solid substrate using a method known in the art (e.g.,semi-dry blotting or tank blotting); contacting the membrane or othersolid substrate to be tested for the presence of bound hRSV F protein ora fragment thereof with an anti-hRSV antibody or antigen-bindingfragment thereof of the invention.

Such a membrane may take the form of a nitrocellulose or vinyl-based(e.g., polyvinylidene fluoride (PVDF)) membrane to which the proteins tobe tested for the presence of hRSV in a non-denaturing PAGE(polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) gel have been transferred(e.g., following electrophoretic separation in the gel). Beforecontacting the membrane with the anti-hRSV antibody or fragment, themembrane is optionally blocked, e.g., with non-fat dry milk or the likeso as to bind non-specific protein binding sites on the membrane.

(2) washing the membrane one or more times to remove unbound anti-hRSV Fprotein antibody or fragment and other unbound substances; and

(3) detecting the bound anti-hRSV F protein antibody or fragment.

Detection of the bound antibody or fragment indicates that the hRSVprotein is present on the membrane or substrate and in the sample.Detection of the bound antibody or fragment may be by binding theantibody or fragment with a secondary antibody (an anti-immunoglobulinantibody) which is detectably labeled and, then, detecting the presenceof the secondary antibody.

The anti-hRSV F protein antibodies and antigen-binding fragments thereofdisclosed herein (e.g., RB1) may also be used for immunohistochemistry.Such a method forms part of the present invention and comprises, e.g.,

(1) contacting a cell to be tested for the presence of hRSV F proteinwith an anti-hRSV F protein antibody or antigen-binding fragment thereofof the invention; and

(2) detecting the antibody or fragment on or in the cell.

If the antibody or fragment itself is detectably labeled, it can bedetected directly. Alternatively, the antibody or fragment may be boundby a detectably labeled secondary antibody which is detected.

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the anti-hRSV Fprotein antibodies and antigen-binding fragments of the invention (e.g.,RB1), the antibody or antigen-binding fragment thereof is admixed with apharmaceutically acceptable carrier or excipient. See, e.g., Remington'sPharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, MackPublishing Company, Easton, Pa. (1984).

Formulations of therapeutic and diagnostic agents may be prepared bymixing with acceptable carriers, excipients, or stabilizers in the formof, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.). In one embodiment, antibodies or antigen bindingfragments thereof of the present invention are diluted to an appropriateconcentration in a histidine buffer pH 5-7, at 1-20 mM and NaCl orsucrose (e.g., 2-15% (w/v)) is optionally added for tonicity. Additionalagents, such as polysorbate 20 or polysorbate 80, at 0.01 to 0.10% (w/v)may be added to enhance stability. A representative formulation is 10 mML-Histidine, 7% (w/v) Sucrose, and 0.02% (w/v) polysorbate-80, pH 6.0.

Toxicity and therapeutic efficacy of the antibodies or antigen bindingfragments thereof of the invention, administered alone or in combinationwith another therapeutic agent, can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index (LD₅₀/ED₅₀). The data obtained from these cellculture assays and animal studies can be used in formulating a range ofdosage for use in human. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration.

The mode of administration can vary. Routes of administration includeoral, rectal, transmucosal, intestinal, parenteral; intramuscular,subcutaneous, intradermal, intramedullary, intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, intraocular,inhalation, insufflation, topical, cutaneous, transdermal, orintra-arterial. Preferred modes of administration are intramuscular,intravenous and intranasal.

In particular embodiments, the anti-hRSV F protein antibodies orantigen-binding fragments thereof of the invention (e.g., RB1) can beadministered by an invasive route such as by injection. In furtherembodiments of the invention, an anti-hRSV F protein antibody orantigen-binding fragment thereof, or pharmaceutical composition thereof,is administered intravenously, subcutaneously, intramuscularly,intraarterially, intratumorally, or by inhalation, aerosol delivery.Administration by non-invasive routes (e.g., orally; for example, in apill, capsule or tablet) is also within the scope of the presentinvention.

The present invention provides a vessel (e.g., a plastic or glass vial,e.g., with a cap or a chromatography column, hollow bore needle or asyringe cylinder) comprising any of the antibodies or antigen-bindingfragments of the invention (e.g., RB1) or a pharmaceutical compositionthereof. The present invention also provides an injection devicecomprising any of the antibodies or antigen-binding fragments of theinvention (e.g., RB1) or a pharmaceutical composition thereof. Aninjection device is a device that introduces a substance into the bodyof a patient via a parenteral route, e.g., intramuscular, subcutaneousor intravenous. For example, an injection device may be a syringe (e.g.,pre-filled with the pharmaceutical composition, such as anauto-injector) which, for example, includes a cylinder or barrel forholding fluid to be injected (e.g., antibody or fragment or apharmaceutical composition thereof), a needle for piecing skin and/orblood vessels for injection of the fluid; and a plunger for pushing thefluid out of the cylinder and through the needle bore. In an embodimentof the invention, an injection device that comprises an antibody orantigen-binding fragment thereof of the present invention or apharmaceutical composition thereof is an intravenous (IV) injectiondevice. Such a device includes the antibody or fragment or apharmaceutical composition thereof in a cannula or trocar/needle whichmay be attached to a tube which may be attached to a bag or reservoirfor holding fluid (e.g., saline; or lactated ringer solution comprisingNaCl, sodium lactate, KCl, CaCl₂ and optionally including glucose)introduced into the body of the patient through the cannula ortrocar/needle. The antibody or fragment or a pharmaceutical compositionthereof may, in an embodiment of the invention, be introduced into thedevice once the trocar and cannula are inserted into the vein of asubject and the trocar is removed from the inserted cannula. The IVdevice may, for example, be inserted into a peripheral vein (e.g., inthe hand or arm); the superior vena cava or inferior vena cava, orwithin the right atrium of the heart (e.g., a central IV); or into asubclavian, internal jugular, or a femoral vein and, for example,advanced toward the heart until it reaches the superior vena cava orright atrium (e.g., a central venous line). In an embodiment of theinvention, an injection device is an autoinjector; a jet injector or anexternal infusion pump. A jet injector uses a high-pressure narrow jetof liquid which penetrate the epidermis to introduce the antibody orfragment or a pharmaceutical composition thereof to a patient's body.External infusion pumps are medical devices that deliver the antibody orfragment or a pharmaceutical composition thereof into a patient's bodyin controlled amounts. External infusion pumps may be poweredelectrically or mechanically. Different pumps operate in different ways,for example, a syringe pump holds fluid in the reservoir of a syringe,and a moveable piston controls fluid delivery, an elastomeric pump holdsfluid in a stretchable balloon reservoir, and pressure from the elasticwalls of the balloon drives fluid delivery. In a peristaltic pump, a setof rollers pinches down on a length of flexible tubing, pushing fluidforward. In a multi-channel pump, fluids can be delivered from multiplereservoirs at multiple rates.

The pharmaceutical compositions disclosed herein may also beadministered with a needleless hypodermic injection device; such as thedevices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Suchneedleless devices comprising the pharmaceutical composition are alsopart of the present invention. The pharmaceutical compositions disclosedherein may also be administered by infusion. Examples of well-knownimplants and modules for administering the pharmaceutical compositionsinclude those disclosed in: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments. Many other such implants, delivery systems, and modulesare well known to those skilled in the art and those comprising thepharmaceutical compositions of the present invention are within thescope of the present invention.

The administration regimen depends on several factors, including theserum or tissue turnover rate of the therapeutic antibody orantigen-binding fragment, the level of symptoms, the immunogenicity ofthe prophylactic/therapeutic antibody, and the accessibility of thetarget cells in the biological matrix. Preferably, the administrationregimen delivers sufficient therapeutic antibody or fragment to effectimprovement in the target disease state, while simultaneously minimizingundesired side effects. Accordingly, the amount of biologic delivereddepends in part on the particular prophylactic/therapeutic antibody andthe severity of the condition being treated. Guidance in selectingappropriate doses of therapeutic antibodies or fragments is available(see, e.g., Wawrzynczak, (1996) Antibody Therapy, Bios Scientific Pub.Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies,Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.)(1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases,Marcel Dekker, New York, N.Y.; Baert, et al., 2003, New Engl. J. Med.348:601-608; Milgrom et al., 1999, New Engl. J. Med. 341:1966-1973;Slamon et al., 2001, New Engl. J. Med 344:783-792; Beniaminovitz et al.,2000, New Engl. J. Med. 342:613-619; Ghosh et al., 2003, New Engl. J.Med. 348:24-32; Lipsky et al., 2000, New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affectprevention or treatment. Generally, the dose begins with an amountsomewhat less than the optimum dose and it is increased by smallincrements thereafter until the desired or optimum effect is achievedrelative to any negative side effects. Important diagnostic measuresinclude those of symptoms of, e.g., the inflammation or level ofinflammatory cytokines produced. In general, it is desirable that abiologic that will be used is derived from the same species as theanimal targeted for treatment, thereby minimizing any immune response tothe reagent. In the case of human subjects, for example, humanized andfully human antibodies are may be desirable.

Antibodies or antigen-binding fragments thereof disclosed herein (e.g.,RB1) may be provided by continuous infusion, or by doses administered,e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly,quarterly, semiannually, annually etc. Doses may be provided, e.g.,intravenously, subcutaneously, topically, orally, nasally, rectally,intramuscular, intracerebrally, intraspinally, or by inhalation. A totalweekly dose is generally at least 0.05 μg/kg body weight, more generallyat least 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg,1.0 mg/kg, 2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more(see, e.g., Yang, et al., 2003, New Engl. J. Med. 349:427-434; Herold,et al., 2002, New Engl. J. Med. 346:1692-1698; Liu, et al., 1999, J.Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al., 2003, CancerImmunol. Immunother. 52:151-144). Doses may also be provided to achievea pre-determined target concentration of anti-hRSV antibody in thesubject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/ml or more.In other embodiments, an anti-hRSV antibody of the present invention isadministered, e.g., subcutaneously or intravenously, on a weekly,biweekly, “every 4 weeks,” monthly, bimonthly, or quarterly basis at 10,20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.

As used herein, the term “effective amount” refer to an amount of ananti-hRSV or antigen-binding fragment thereof of the invention (e.g.,RB1) that, when administered alone or in combination with an additionaltherapeutic/prophylactic agent to a cell, tissue, or subject, iseffective to neutralize RSV and/or prevent or cause a measurableimprovement in one or more symptoms of disease or condition associatedwith RSV infection. An effective dose further refers to that amount ofthe antibody or fragment sufficient to result in at least partialprevention or amelioration of symptoms. When applied to an individualactive ingredient administered alone, an effective dose refers to thatingredient alone. When applied to a combination, an effective doserefers to combined amounts of the active ingredients that result in theprophylactic or therapeutic effect, whether administered in combination,serially or simultaneously. In certain embodiments, an effective amountis an amount that provides a clinical target serum concentration of 10μg/mL-30 μg/mL for 5 months. In one embodiment, an effective amount is ahuman dose that provides a Ctrough target of 10 μg/ml-30 μg/ml forefficacy, as determined in standard pre-clinical cotton rat models.

Kits

Further provided are kits comprising one or more components thatinclude, but are not limited to, an anti-hRSV F protein antibody orantigen-binding fragment, as discussed herein (e.g., RB1) in associationwith one or more additional components including, but not limited to apharmaceutically acceptable carrier and/or a prophylactic/therapeuticagent, as discussed herein. The antibody or fragment and/or theprophylactic/therapeutic agent can be formulated as a pure compositionor in combination with a pharmaceutically acceptable carrier, in apharmaceutical composition.

In one embodiment, the kit includes an anti-hRSV F protein antibody orantigen-binding fragment thereof of the invention (e.g., RB1) or apharmaceutical composition thereof in one container (e.g., in a sterileglass or plastic vial) and a pharmaceutical composition thereof and/or aprophylactic/therapeutic agent in another container (e.g., in a sterileglass or plastic vial).

In another embodiment, the kit comprises a combination of the invention,including an anti-hRSV F protein antibody or antigen-binding fragmentthereof of the invention (e.g., RB1) along with a pharmaceuticallyacceptable carrier, optionally in combination with one or moreprophylactic/therapeutic agents formulated together, optionally, in apharmaceutical composition, in a single, common container.

If the kit includes a pharmaceutical composition for parenteraladministration to a subject, the kit can include a device for performingsuch administration. For example, the kit can include one or morehypodermic needles or other injection devices as discussed above.

The kit can include a package insert including information concerningthe pharmaceutical compositions and dosage forms in the kit. Generally,such information aids patients and physicians in using the enclosedpharmaceutical compositions and dosage forms effectively and safely. Forexample, the following information regarding a combination of theinvention may be supplied in the insert: pharmacokinetics,pharmacodynamics, clinical studies, efficacy parameters, indications andusage, contraindications, warnings, precautions, adverse reactions,overdosage, proper dosage and administration, how supplied, properstorage conditions, references, manufacturer/distributor information andpatent information.

Detection Kits and Prophylactic/Therapeutic Kits

As a matter of convenience, an anti-hRSV antibody or antigen-bindingfragment thereof of the invention (e.g., RB1) can be provided in a kit,i.e., a packaged combination of reagents in predetermined amounts withinstructions for performing a diagnostic or detection assay. Where theantibody or fragment is labeled with an enzyme, the kit will includesubstrates and cofactors required by the enzyme (e.g., a substrateprecursor which provides the detectable chromophore or fluorophore). Inaddition, other additives may be included such as stabilizers, buffers(e.g., a block buffer or lysis buffer) and the like. The relativeamounts of the various reagents may be varied widely to provide forconcentrations in solution of the reagents which substantially optimizethe sensitivity of the assay. Particularly, the reagents may be providedas dry powders, usually lyophilized, including excipients which ondissolution will provide a reagent solution having the appropriateconcentration.

Also provided are diagnostic or detection reagents and kits comprisingone or more such reagents for use in a variety of detection assays,including for example, immunoassays such as ELISA (sandwich-type orcompetitive format). The kit's components may be pre-attached to a solidsupport, or may be applied to the surface of a solid support when thekit is used. In some embodiments of the invention, the signal generatingmeans may come pre-associated with an antibody or fragment of theinvention or may require combination with one or more components, e.g.,buffers, antibody-enzyme conjugates, enzyme substrates, or the like,prior to use. Kits may also include additional reagents, e.g., blockingreagents for reducing nonspecific binding to the solid phase surface,washing reagents, enzyme substrates, and the like. The solid phasesurface may be in the form of a tube, a bead, a microtiter plate, amicrosphere, or other materials suitable for immobilizing proteins,peptides, or polypeptides. In particular aspects, an enzyme thatcatalyzes the formation of a chemilluminescent or chromogenic product orthe reduction of a chemilluminescent or chromogenic substrate is acomponent of the signal generating means. Such enzymes are well known inthe art. Kits may comprise any of the capture agents and detectionreagents described herein. Optionally the kit may also compriseinstructions for carrying out the methods of the invention.

Also provided is a kit comprising an anti-hRSV F protein antibody orantigen-binding fragment thereof packaged in a container, such as a vialor bottle, and further comprising a label attached to or packaged withthe container, the label describing the contents of the container andproviding indications and/or instructions regarding use of the contentsof the container to prevent/treat one or more disease states asdescribed herein.

In one aspect, the kit is for preventing or treating diseases/conditionsassociated with RSV infection and comprises an anti-hRSV F proteinantibody or antigen-binding fragment thereof and a furtherprophylactic/therapeutic agent or a vaccine. The kit may optionallyfurther include a syringe for parenteral, e.g., intravenous,administration. In another aspect, the kit comprises an anti-hRSV Fprotein antibody or antigen-binding fragment thereof and a labelattached to or packaged with the container describing use of theantibody or fragment with the vaccine or furtherprophylactic/therapeutic agent. In yet another aspect, the kit comprisesthe vaccine or further prophylactic/therapeutic agent and a labelattached to or packaged with the container describing use of the vaccineor further prophylactic/therapeutic agent with the anti-hRSV F proteinantibody or fragment. In certain embodiments, an anti-hRSV F proteinantibody and vaccine or further prophylactic/therapeutic agent are inseparate vials or are combined together in the same pharmaceuticalcomposition.

For combination prophylaxis or therapy, concurrent administration of twoprophylactic/therapeutic agents does not require that the agents beadministered at the same time or by the same route, as long as there isan overlap in the time period during which the agents are exerting theirprophylactic/therapeutic effect. Simultaneous or sequentialadministration is contemplated, as is administration on different daysor weeks.

The prophylactic/therapeutic and detection kits disclosed herein mayalso be prepared that comprise at least one of the antibody, peptide,antigen-binding fragment, or polynucleotide disclosed herein andinstructions for using the composition as a detection reagent orprophylactic/therapeutic agent. Containers for use in such kits maytypically comprise at least one vial, test tube, flask, bottle, syringeor other suitable container, into which one or more of the detectionand/or prophylactic/therapeutic composition(s) may be placed, andpreferably suitably aliquoted. Where a second prophylactic/therapeuticagent is also provided, the kit may also contain a second distinctcontainer into which this second detection and/orprophylactic/therapeutic composition may be placed. Alternatively, aplurality of compounds may be prepared in a single pharmaceuticalcomposition, and may be packaged in a single container means, such as avial, flask, syringe, bottle, or other suitable single container. Thekits disclosed herein will also typically include a means for containingthe vial(s) in close confinement for commercial sale, such as, e.g.,injection or blow-molded plastic containers into which the desiredvial(s) are retained. Where a radiolabel, chromogenic, fluorigenic, orother type of detectable label or detecting means is included within thekit, the labeling agent may be provided either in the same container asthe detection or prophylactic/therapeutic composition itself, or mayalternatively be placed in a second distinct container means into whichthis second composition may be placed and suitably aliquoted.Alternatively, the detection reagent and the label may be prepared in asingle container means, and in most cases, the kit will also typicallyinclude a means for containing the vial(s) in close confinement forcommercial sale and/or convenient packaging and delivery.

A device or apparatus for carrying out the detection or monitoringmethods described herein is also provided. Such an apparatus may includea chamber or tube into which sample can be input, a fluid handlingsystem optionally including valves or pumps to direct flow of the samplethrough the device, optionally filters to separate plasma or serum fromblood, mixing chambers for the addition of capture agents or detectionreagents, and optionally a detection device for detecting the amount ofdetectable label bound to the capture agent immunocomplex. The flow ofsample may be passive (e.g., by capillary, hydrostatic, or other forcesthat do not require further manipulation of the device once sample isapplied) or active (e.g., by application of force generated viamechanical pumps, electroosmotic pumps, centrifugal force, or increasedair pressure), or by a combination of active and passive forces.

In further embodiments, also provided is a processor, a computerreadable memory, and a routine stored on the computer readable memoryand adapted to be executed on the processor to perform any of themethods described herein. Examples of suitable computing systems,environments, and/or configurations include personal computers, servercomputers, hand-held or laptop devices, multiprocessor systems,microprocessor-based systems, set top boxes, programmable consumerelectronics, network PCs, minicomputers, mainframe computers,distributed computing environments that include any of the above systemsor devices, or any other systems known in the art.

GENERAL METHODS

Standard methods in molecular biology are described Sambrook, Fritschand Maniatis (1982, 1989 2^(nd) Edition, and 2001 3^(rd) Edition)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001) MolecularCloning, 3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, SanDiego, Calif.). Standard methods also appear in Ausbel et al. (2001)Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons,Inc. New York, N.Y., which describes cloning in bacterial cells and DNAmutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2),glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,N.Y., pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production,purification, and fragmentation of polyclonal and monoclonal antibodiesare described (Coligan, et al. (2001) Current Protocols in Immunology,Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, JohnWiley, Inc., New York).

Single chain antibodies and diabodies are described (see, e.g., Maleckiet al., 2002, Proc. Natl. Acad. Sci. USA 99:213-218; Conrath et al.,2001, J Biol. Chem. 276:7346-7350; Desmyter et al., 2001, J. Biol. Chem.276:26285-26290; Hudson and Kortt, 1999, J. Immunol. Methods231:177-189; and U.S. Pat. No. 4,946,778). Bifunctional antibodies areprovided (see, e.g., Mack, et al. (1995) Proc. Natl. Acad. Sci. USA92:7021-7025; Carter (2001) J. Immunol. Methods 248:7-15; Volkel, et al.(2001) Protein Engineering 14:815-823; Segal, et al. (2001)J. Immunol.Methods 248:1-6; Brennan, et al. (1985) Science 229:81-83; Raso, et al.(1997) J. Biol. Chem. 272:27623; Morrison (1985) Science 229:1202-1207;Traunecker, et al. (1991) EMBO J. 10:3655-3659; and U.S. Pat. Nos.5,932,448, 5,532,210, and 6,129,914).

Bispecific antibodies are also provided (see, e.g., Azzoni et al. (1998)J. Immunol. 161:3493; Kita et al. (1999) J. Immunol. 162:6901; Merchantet al. (2000) J. Biol. Chem. 74:9115; Pandey et al. (2000) J. Biol.Chem. 275:38633; Zheng et al. (2001) J. Biol Chem. 276:12999; Propst etal. (2000) J. Immunol. 165:2214; Long (1999) Ann. Rev. Immunol. 17:875).

Antibodies can be conjugated, e.g., to small drug molecules, enzymes,liposomes, polyethylene glycol (PEG). Antibodies are useful fortherapeutic, diagnostic, kit or other purposes, and include antibodiescoupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g.,colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol.146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsingand Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002)J.Immunol. 168:883-889).

Methods for flow cytometry, including fluorescence activated cellsorting (FACS), are available (see, e.g., Owens, et al. (1994) FlowCytometry Principles for Clinical Laboratory Practice, John Wiley andSons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd) ed.;Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, JohnWiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable (Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne, et al. (2000) Bioinformatics 16: 741-742;Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren,et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690).

EXAMPLES Example 1: Identification of a Fully Human RSV NeutralizingAntibody

In order to identify potent HRSV neutralizing antibodies, serum wasobtained from donors under informed consent and assayed for the abilityto neutralize HRSV virus in vitro. For the neutralization assay, serumsamples were first serially diluted and then incubated with 600 pfu of ahRSV-A strain expressing the enhanced green fluorescent protein(RSV-GFP). The RSV-GFP was mixed 1:1 with serum dilutions in a totalvolume of 200 μl per well in 96-well U-bottom plates at 37° C. for 1 hr.100 μl of the mixture per well was then transferred to HEp-2 cell seededplates (15,000 cells per well). The plates were scanned on acumen®Cellista (TTP LabTech, Cambridge, Mass.) and data were exported asnumber of GFP events and total fluorescence intensity per well. NT50values were calculated using GraphPad Prism 6 (GraphPad Software, Inc.,La Jolla, Calif.) by four parameter curve fitting. ELISA binding titersto RSV pre-F and post-F proteins were performed as per the following.Nunc C96 Maxisorp® Nunc-Immuno™ plates (Thermo Scientific, Inc.) werecoated with 50 μl per well of hRSV pre-F (See McLellan et al., 2013,Science 342:592) or post-F protein (post-fusion F LZF21 protein consistsof the wt F ectodomain without the fusion peptide (See McLellen et al.,2011, J. Virol 85:7788)) at 1 μg/ml in PBS at 4° C. overnight. Theplates were washed with PBS/Tween 20 and then blocked with 3% non-fatmilk in PBS. Afterwards, 50 μl of serially diluted serum samples wereadded per well and incubated at room temperature for 90 min. The plateswere washed and HRP-conjugated goat anti-human IgG (SouthernBiotech,Birmingham, Ala.) was added at 1:2,000 dilution. One hour later, theplates were washed and developed with SuperBlu Turbo TMB solution(ViroLabs, Inc., Sterling, Va.). OD450nm readings were obtained using aWallac 1420 VICTOR²™ Multilabel Counter (Perkin Elmer, Waltham, Mass.).EC50 values were calculated using GraphPad Prism 6 by four parametercurve fitting. A subset of the donors that demonstrated high HRSVneutralizing and binding titers were recalled to procure larger bloodvolumes for PBMC generation. PBMC preparation was carried out by acommercial vendor and the purchased PBMC's were stored at liquidnitrogen until further use.

PBMC's from one subject demonstrated good neutralization titers and hadalso the highest titers in a ELISA binding assay to Post Fusion Fprotein as well as being one of the elite binders to pre-Fusion Fprotein (data not shown). Therefore, this donor's PBMCs were chosen toisolate Post-F specific memory B-cells by FACS sorting.

Biotinylated trimeric post-fusion F protein (LZF21) was prepared bybiotinylating LZF21 (McLellen et al., 2011, J. Virol 85:7788) using E-ZLink™ Sulfo-NHS-LC biotinylation kit (Life Technologies, Grand Island,N.Y.) according to the manufacturer's instructions. The LZF21 proteinconsists of the wild-type F protein ectodomain without the fusionpeptide (McLellen et al., 2011, J. Virol 85:7788). The fusion F proteinspecific murine hybridoma 4D7 (4D7 is a mouse hybridoma that wasgenerated by immunizing Balb/c mice with RSV A2 virus). Balb/c mice wereimmunized twice, intraperitoneally, with RSV A2 virus and boosted threedays prior to fusion, using 20 μg of purified RSV A2 (AdvancedBiotechnologies, Inc., Columbia, Md.), by intravenous injection. Thespleen was harvested and splenocytes were fused with SP2/0 myeloma cellsusing polyethylene glycol. Cells were added to Medium D (StemCellTechnologies Inc., Vancouver, BC), plated in 245 mm×245 mm square petridishes and incubated at 37° C., 5% CO₂ for 2 weeks. Individual colonieswere picked using a ClonePix (Genetix), transferred to 96 well platesand incubated as above for 1 week. Supernatants were then screened foranti-RSV activity by ELISA against purified RSV A2. Positive clones,including 4D7, were expanded and further sub-cloned by limitingdilution. Sub-clones were screened as described above and 4D7-8 wasidentified and used to optimize the staining of LZF21-biotin to memoryB-cells by FACS (data not shown). Through these optimizationexperiments, it was determined that 1.5 μg/ml of LZF21-biotin was thebest concentration for staining of post-F specific memory B-cells. Thespecificity of the staining reaction was demonstrated by using anirrelevant murine hybridoma as a negative control and competing thebinding with 100-1000× excess of unlabeled LZF21.

Antigen specific memory B-cells were delineated as CD3⁻CD19⁺IgG⁺LZF21⁺.These cells were sorted into a 96 well plates (one cell/well) containinga CD40 ligand expressing HEK293 cell line (made using standard molecularbiology techniques) and IL-21 (Sino Biological Inc., North Wales, Pa.).Out of 30 sorted samples, the supernatant from 6 wells demonstratedbinding to post-F protein in an ELISA assay (performed as describedabove). These samples also bound to pre-F protein (data not shown).These six samples were then tested in neutralization assay, as describedabove, without dilution. The presence of neutralization activity wasdetermined based on the reduction of GFP events. Among six samples, twoof them (designated RB1 and RB11) showed complete neutralization ofHRSV-A strain (See Table 3).

TABLE 3 ELISA 450 nm Neutralization Well ID IgG Post F Pre-F GFP Object#% Neut A8 0.641 1.743 1.222 783 A9 1.743 1.915 1.754 689 A11 1.81 1.9051.555 500 B1 1.82 1.925 1.910 0 100% B11 1.851 1.748 1.900 1 100% B121.801 1.838 1.679 548 Control 0.037 0.038 0.044 596

RNA Extraction and RT-PCT For Single-Sorted Memory B Cell Culture

Part 1:

The RNA from a 96 well plate from RB1 hit lysate was extracted using aRNeasy® Micro Kit (Qiagen, Inc., Valencia, Calif.) as per themanufacturer's manual. The RNA concentration was determined withNanoDrop™ 2000C (Thermo Fisher Scientific Inc., Wilmington, Del.) underUV 260 nm. The extracted RNA from the RB1 well was used as template inthe RT-PCR amplification of antibody heavy and light chain genes usingprimer sequences using sequences from the leader sequence (forward) andC′ end of IgG JH, Kappa constant region or Lambda constant region(reverse).

OneStep RT-PCR kit (Qiagen Inc, Valencia, Calif.) was used according tothe manufacturer's instructions to amplify the antibody sequences. ThePCR conditions were as follows: 50° C. for 30 mins, 95° C. for 15 mins,[94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 1 min]×40, 72° C. for10 min, and 4° C. hold.

The RT-PCR product was directly used as a template for nested PCR.

Part 2: Nested PCR

The RT-PCR products were used as templates in nested-PCR to amplifyantibody variable regions with pfx50 DNA polymerase (Invitrogen, catalogno: 12355-012). The design for nested-PCR primers was based on germlinesequences of framework 1 region of human IgG heavy and light chainvariable regions.

1 μl RT-PCR product was mixed with 2.5 μl 10×PCR buffer, 2.5 μl 10×PCRXEnhancer (Invitrogen), 0.5 μl dNTP mix, 0.5 μl forward primer pool (10μM each), 0.5 μl reverse primer (10 μM), 0.5 μl pfx50 DNA polymerase and17 μl water. Nested PCR condition was: 2 min at 94° C., 10 cycles of 94°C. for 30 sec, 50° C. for 30 sec, 68° C. for 1 min, followed by 30cycles of 94° C. for 30 sec, 60° C. for 30 sec, 68° C. for 1 min, then 7min elongation at 68C, followed by 4° C. hold for short term storage.

The RB1 nested PCR products of VH and VK or VH and VL amplified PCRproducts were used as template in the overlap PCR with specific linkersfor annealing antibody light and heavy chain genes together tofacilitate the next step infusion cloning.

Part 3. Overlap PCR and Infusion Cloning

pfx50 DNA polymerase (Invitrogen) was used in this reaction. Forward andreverse primers were designed to facilitate the infusion cloning ofoverlap PCR products into a cloning vector. 1 μl heavy chain nested PCRproduct, 1 μl light chain nested PCR product and 1 μl linker were mixedwith 5 μl 10×PCR buffer, 5 μl 10×PCRX enhancer, 1 μl dNTP mix, 1 μlforward primer (10 μM), 1 μl reverse primer (10 μM), 1 μl pfx50 DNApolymerase and 33 μl water.

PCR conditions were as follows: 94° C. for 2 mins, [94° C. for 30 sec,60° C. for 30 sec, 68° C. for 2 min]×10, [94° C. for 30 sec, 65° C. for30 sec, 68° C. for 2 min]×30, 68° C. for 7 min, 4° C. hold.

The overlap PCR products were agarose gel purified for infusion cloning(a RB1 VH+VK overlap PCR product of around 1.2 kb was obtained). The RB1VH+VK overlap PCR products were cloned into pMab11Exp2 (with OmpA leadersequence for light chain expression, with PelB leader sequence for heavychain expression) vector with the application of infusion cloning.Infusion HD® cloning Kit (Clontech Laboratories, Inc., Mountain View,Calif.) was used and the manufacturer's instructions were followed.Transformants were picked and sent to GeneWiz, Inc. (South Plainfield,N.J.) for sequencing.

The sequencing results were analyzed with Sequencher (Gene CodesCorporation, Ann Arbor, Mich.).

The nucleotide and amino acid sequences of RB1 are depicted in Table 7(the patient isolated RB1 variable heavy chain and variable light chainamino acid sequences are represented by SEQ ID NO: 9 and SEQ ID NO: 8,respectively). Due to the design of the Jh reverse primers having anucleotide change, an isoleucine present in the natural sequence atposition 125 was changed to threonine in the expressed protein (theresulting RB1 variable heavy chain is represented by SEQ ID NO: 7).Amino acid sequences of RB1 antibody heavy and light chain variabledomain genes were sent to GenScript USA, Inc. (Piscataway, N.J.) forcodon optimization and human IgG1 conversion and CHO transientexpression and production. Synthesized DNAs were subcloned into pTT5vector for CHO-3E7 cell expression. The recombinant plasmids encodingheavy and light chains of each antibody were transiently co-transfectedinto CHO-3E7 cell cultures. The cell culture supernatants collected onday 6 were used for purification through Protein A column. Purified RB1human IgG1 was used in neutralization assay and other characterizationexperiments as described in Example 2.

Example 2: Characterization of Anti-hRSV Antibodies

RB1 bound to both Pre F and post fusion F protein in an ELISA assay asdescribed in Example 1 with an EC₅₀ ranging from 1-10 ng/ml, whereas theD25 antibody (See Kwakkenbos et al., 2010, Nature Medicine 16: 123-128)bound preferentially to pre-fusion F. See FIGS. 1A-B.

mAb ID Pre-F (EC50 ng/ml) Post-F (EC50 ng/ml) D25 8.939 >10,000palivizumab 17.37 10.5 RB1 7.053 14.08

Neutralization for RB1, RB 11, and some benchmark antibodies reported inthe literature (D25, palivizumab, full length [D25 antibody was made inhouse based on the published sequence and SYNAGIS® (palivizumab) waspurchased from Myoderm, Norristown, Pa.]) was compared in RSV A Longstrain (ATCC Number VR-26™) and RSV B Washington strain18537 strain(ATCC Number VR-1580TH). The test samples were three-fold seriallydiluted in EMEM supplemented with 2% heat inactivated FBS, for elevendilution points. The serially diluted samples were then mixed with equalvolumes of EMEM supplemented with 2% heat inactivated FBS containing 100pfu/well of RSV A or B strains. After incubation at 37° C. for 1 hr, 100μl of HEp-2 cells at a concentration of 1.5×10⁵ cells/ml was transferredto the 96 well plates containing the virus/antibody mixture. At 3 dayspost infection, the cells were washed once with PBS and then fixed in80% acetone for 10 min at room temperature. A mixture of RSV F(mAb143-F3-B138) and RSV N (34C9) specific mouse mAbs (obtainedin-house) was added to the plates and incubated for 1 hour at roomtemperature. Plates were washed with PBS/0.05% Tween 20 and biotinylatedhorse anti-mouse IgG was added to the plates and incubated for 1 hour atroom temperature. Plates were washed with PBS/0.05% Tween. Infrareddye-Streptavidin was used to detect RSV specific signal and two cellstains for assay normalization were added to the 96-well plates andincubated for 1 hour in the dark. Following 1 hour incubation, theplates were washed, air dried for 20 minutes in the dark and read on theLicor Aerius® Automated Imaging System utilizing a 700 channel laser forcell normalization and an 800 channel laser for detection of RSVspecific signal. 800/700 ratios and percent neutralization werecalculated and IC50 values were determined by four parameter curve fitin GraphPad.

RB1 was able to neutralize the RSV-A and RSV-B strains with equalpotency (IC50 of 1-5 ng/ml). RB1 also demonstrated superior anti-RSVneutralization compared to the benchmark antibodies. See FIGS. 2A-B andTable 4.

TABLE 4 Binding and neutralization potency of RB1 compared to benchmarkantibodies Neutralizing IC50 (ng/mL) Pre-fusion Post-fusion F Activity,RSV F binding binding RSV A/Long B/washington RB1 + + 3 1.7 D25 + − 3.625.9 AM22 + − 50 172.8 131-2A, mouse +/− + 1046 >10,000 (Millipore) 4D7(Merck), +/− + 2408 >10,000 mouse palivizumab + + 211.5 166 MPE8 + −106.6 46 101F, mouse + + 67 43.6 AM14 + − 3.2 1.9

Affinity determination for binding of RB1 for pre- and post-fusion Fprotein: The kinetic binding activity of anti-human RSV F proteinantibody RB1 (made as described in Example 1) was measured by surfaceplasmon resonance using a Biacore T200 system (Biacore, GE Healthcare,Piscataway, N.J.). Approximately 5000 RU of Anti-mouse IgG, GEHealthcare Catalog Number BR-1008-38, or approximately 13,000 RU of GoatAnti-Rat IgG Fc gamma, Fragment Specific, Jackson ImmunoResearch CatalogNumber 112-006-071, was immobilized via amine coupling chemistry onto aSeries S CM5 sensor chip, catalog number BR-1005-30.

Background subtraction binding sensorgrams were used for analyzing therate constant of association (k_(a)) and dissociation (k_(d)), and theequilibrium dissociation constant K_(D). The resulting data sets werefitted with a 1:1 Langmuir Binding Model using the Biacore T200evaluation software (version 2.0). Table 5 summarizes the affinities forthe anti-human RSV F protein antibody to the pre-fusion and post-fusionforms of the RSV F protein.

TABLE 5 Measurement of Affinity for RB1 to RSV pre-fusion F andpost-fusion F using BIAcore Protein K_(on) (M-1S-1) K^(off) (S-1) K_(D)(nM) pre-Fusion F 4.4 × 10⁶ 1.4 × 10⁻⁴ 0.031 Post-Fusion F 2.2 × 10⁶   9× 10⁻⁴ 0.41

RB1 is a very potent binder of pre-fusion F protein with a Kd of ˜31 pM.The Kd for post fusion binding was a magnitude lower at 0.41 nM. The Kdfor D25 as reported in International Patent Application Publication No.WO2014121021 Al was 57 pM. Also the antibody RB1 stays on longer onpre-fusion F than post as seen with a slower off rates of 1.4×10⁻⁴ ascompared to post Fusion F protein.

Example 3: Epitope Mapping of RB1 Antibody

RB1's binding epitope on fusion F protein was mapped by carrying out analanine scan mutagenesis experiment. Epitope mapping was performed byshotgun mutagenesis at Integral Molecular as described. (Davidson andDoranz, 2014, Immunology 143(1): 13-20). To construct a shotgunmutagenesis library, RSV-F protein expression vector is mutagenized tocreate a library of clones, each representing an individual point mutantand cumulatively covering all residues in the protein. Libraries wereconstructed using alanine scanning mutagenesis which provides a morecontrolled method of defining the side-chain contributions of eachresidue. Using semi-automated robotic protocols, each mutated plasmidwas individually cloned, sequenced, mini-prepped and arrayed in 384-wellmicroplate format for repeated transfection, expression and antibodybinding assays in human cells. Alanine scanning mutant library wasscreened against RB1 for loss of antibody binding. Two residuesarginine-429 and Isoleucine-432 were identified as critical for RB1binding. See below and FIG. 3A. RB1 appears to be a site IV mAb(101F-like) and Site IV binding antibodies in the literature have beenreported to bind both pre-fusion and post-fusion F.

Binding Reactivity (% WT) Mutation RB1 MAb RB1 Fab D25 MAb R429A 33.1(27) 5.1 4 385.0 (13) I432A 36.7 (39) 3.6 7 327.8 (161)

We further co-crystalized RB1 Fab with pre-fusion F protein tounderstand better the binding epitope. Diffraction data were collectedfrom crystals at 3.4-3.5 A°. The RB1 antibody binds to the pre-fusion Fprotein through interactions with the CDR loops of both heavy and lightchains. The light chain CDR3 loop interacts with the side chain of Arg429 through the formation of two hydrogen bonds between the carbonyloxygens of Phe 91 and Leu 92 and the guanidino nitrogens of Arg 429.Also on the light chain, Asp 50 and Glu 55 on the CDR2 loop arepositioned to form hydrogen bonds with Asn 426 and Lys 445 of RSV.Extensive interactions are made through the CDR3 loop of the heavy chainof RB1, with Tyr 104 and Tyr 110 forming a surface for van der Waalsinteraction with Ile 432 on RSV. Lys 433 of RSV forms a hydrogen bondwith Asn 107 of the CDR3 loop. From the crystal structure, the lightchain of RB1 also packs against Glu 161 and Ser 182 or the neighboringmonomer of the RSV pre-fusion trimer.

The binding epitope that was identified for RB1 is highly conservedamong 944 of 946 F protein sequences reported in the literature. Thissuggests that resistance to antibodies to this region would be expectedto be low.

Example 4: Anti-RSVActivity of RSV Antibodies in Animal Model

RB1 antibody was compared to D25 and palivizumab for affordingprotection in the cotton rat challenge model. The study includedpalivizumab, D25 and RB1 antibodies given at 2.5 mg/k and serial diluted10 fold to 0.25 mg/mk. In this model of passive immunotherapy, cottonrats were given RB1, D25 or palivizumab at various concentration at d0and challenged with 10⁵ pfu of RSV one day later. The nose and the lungtiters of challenged RSV virus were four days post challenge and used todetermine viral shedding via a plaques assay.

Cotton rat: At least five cotton rats (Sigmodon hispidus), 3-7 weeks oldwith an average body weight of approximately 100 grams were obtainedfrom SAGE Labs (Boyertown, Pa.). Conventional rodent chow and water wereprovided ad libitum.

Antibody Reagents: Palivizumab 100 mg lyophilized (Myoderm, Norristown,Pa.) was formulated in water at 100 mg/ml. The other antibodies wereexpressed and purified in house.

Formulations of Antibody Reagents: The formulation buffers were specificfor each antibody to stabilize the proteins and prevent precipitation.The formulations were as follows: RB1 and D25 were diluted in 1×Phosphate Buffered Saline, pH 7.2. Palivizumab was formulated as permanufacturer suggestion by dissolving in distilled H₂O which wouldeffectively buffer the protein in 25 mM histidine and 1.3 mM glycinepH6.0.

Dosing Solution Preparation, Administration, and Analyses:

Five animals were randomly weighed to determine average weight of thecohorts used. Formulations were prepared about one hour prior toadministration into the animals. Frozen stocks of antibodies were thawedon wet ice for a single thaw. Each antibody was diluted to the properdose concentration to be delivered to each group. On day 0 (initiationof the study) animals that were randomly assigned to each group werelightly sedated with 1-4% isoflurane anesthesia and administered 0.1 mlinto the right quadricep intramuscularly with a 26G syringe and needle.Animals recovered from the effects of the sedation within two minutes.About 24 hours later (+/−2 hours), cotton rats were sedated with 1-4%isoflurane, bled via the retroorbital plexus and then immediately dosedthrough the nares with 0.1 ml of 1×10^(5.5) pfu of RSV A2 or RSV BWashington wild type virus in Williams E medium. Four days postinoculation, animals were sacrificed by CO₂ inhalation and lung (leftlobes) and nasal turbinates were removed and homogenized in 10 volumesof Hanks Balanced Salt Solution (Lonza) containing Sucrose PhosphateGlutamine buffer (SPG) on wet ice. Samples were clarified bycentrifugation at 2000 rpm for 10 minutes, aliquoted, flash frozen, andimmediately stored frozen at −70° C. until tested in plaque assay.

As depicted in FIGS. 4A-D and FIGS. 5A-D, RB1 was able to achieve 2-3log reduction in virus titers for both RSV A and RSV B in lung and noseat a dose of 2.5 mpk dose similar to D25 but better than palivizumabwhich was unable to impact the virus titers in the nose.

Example 5: Fc Engineering of RB1

The neonatal Fc receptor for IgG (FcRn) has been well characterized inthe transfer of passive humoral immunity from a mother to her fetus. Inaddition, throughout life, FcRn protects IgG from degradation, therebyexplaining the long half-life of this class of antibody in the serum.See, e.g., Israel et al., 1996, Immunology 89:573-8. FcRn binds to theFc portion of IgG at a site that is distinct from the binding sites ofthe classical FcγRs or the C1q component of complement. The FcRn-Fcco-crystal structure revealed that FcRn binds to the CH2-CH3 hingeregion of IgG antibodies. A distinguishing characteristic of theIgG-FcRn pathway is obligate pH dependence. IgG-FcRn binding is drivenby acidic pH (6.0) in the lysosome, whereas disassociation occurs at theneutral pH (7.4) of the extracellular environment. Acidification (pH6.0-6.5) in the lysosomes enables the binding of FcRn to the Fc regionof IgG with a low micromolar affinity and protects it from catabolism.The protected FcRn-bound IgG is subsequently shuttled to the cellsurface and released into the extracellular environment. This processprotects antibodies by decreasing their exposure to extracellulardegradation.

The RB1 antibody was subjected to Fc Engineering in an effort to improvehalf-life. RB1+YTE is a derivative of RB1 with the triple mutation(M252Y/S254T/T256E (YTE)) introduced into the Fc portion of RB1. ThisYTE mutation set improves antibody binding to neonatal Fc receptorsleading to longer half lives in humans. See, e.g., Dall'Acqua et al.,2006, J Biol Chem. 281:23514-24. Mutations in 3 amino acids (YTE:M252Y/S254T/T256E) within the Fc region of motavizumab has led to a10-fold increase in in vitro FcRn binding at pH 6.0 for both humans andmonkeys consequently resulting in a 4-fold increase in in vivo serumhalf-life in monkeys. See Robbie et al., 2013, Antimicrob AgentsChemother. 57:6147-53.

Amino acid sequences of RB1+YTE antibody heavy and light chain variabledomains were sent to GenScript USA, Inc. (Piscataway, N.J.) for codonoptimization and human IgG1 conversion and CHO transient expression andproduction. The nucleotide and amino acid sequences of RB1+YTE aredepicted in Table 7.

Synthesized DNAs were subcloned into pTT5 vector for CHO-3E7 cellexpression. The recombinant plasmids encoding heavy and light chains ofeach antibody were transiently co-transfected into CHO-3E7 cellcultures. The cell culture supernatants collected on day 6 were used forpurification through Protein A column. Purified RB1+YTE human IgG1 wasused in a neutralization assay and other characterization experiments.

Example 6: Characterization of RB1+YTE

RB-1+YTE bound to F protein in an ELISA assay performed as described inExample 1 with an EC₅₀ ranging from 1.97 to 2.457 ng/ml. See FIG. 6.Neutralization for RB1+YTE, and a benchmark antibody reported in theliterature (Motavizumab, MedImmune, Gaithersburg, Md.; see U.S. PatentApplication Publication No. US20110158985) made in house based on thepublished sequence, were compared in RSV A Long strain (ATCC NumberVR-26™) and RSV B Washington strain18537 strain (ATCC Number VR-1580TH).The test samples were three-fold serially diluted in EMEM supplementedwith 2% heat inactivated FBS, for eleven dilution points. The seriallydiluted samples were then mixed with equal volumes of EMEM supplementedwith 2% heat inactivated FBS containing 100 pfu/well of RSV A or Bstrains. After incubation at 37° C. for 1 hr, 100 μl of HEp-2 cells at aconcentration of 1.5×10⁵ cells/ml was transferred to the 96 well platescontaining the virus/antibody mixture. At 3 days post infection, thecells were washed once with PBS and then fixed in 80% acetone for 10 minat room temperature. A mixture of RSV F (mAb143-F3-B138) and RSV N(34C9) specific mouse mAbs (mAb143-F3-B138 and 34C9 are in houseantibodies derived by immunizing mice with the respective antigens andimmortalization of B-cells using the hybridoma technology) was added tothe plates and incubated for 1 hour at room temperature. Plates werewashed with PBS/0.05% Tween 20 and biotinylated horse anti-mouse IgG wasadded to the plates and incubated for 1 hour at room temperature. Plateswere washed with PBS/0.05% Tween. Infrared dye-Streptavidin was used todetect RSV specific signal and two cell stains for assay normalizationwere added to the 96-well plates and incubated for 1 hour in the dark.Following 1 hour incubation, the plates were washed, air dried for 20minutes in the dark and read on the Licor Aerius® Automated ImagingSystem utilizing a 700 channel laser for cell normalization and an 800channel laser for detection of RSV specific signal. 800/700 ratios andpercent neutralization were calculated and IC50 values were determinedby four parameter curve fit in GraphPad.

RB-1+YTE was able to neutralize the RSV-A and RSV-B strains with equalpotency (IC50 of 5-10 ng/ml). See Table 6.

TABLE 6 Measurement of Neutralization and Affinity for RB1 + YTE to RSVpre-fusion F and post-fusion F IC₅₀ In vitro neutralization KineticConstants Kinetic Constants (ng/ml) (pre-fusion F) (post-fusion F) MabRSV A RSV B K_(on)(M⁻¹ s⁻¹) K_(off) (s⁻¹) K_(D) (nM) K_(on)(M⁻¹ s⁻¹)K_(off) (s⁻¹) K_(D) (nM) RB-1 3 1.7 4.4 × 10⁶ 1.4 × 10⁻⁴ 0.031 2.2 × 10⁶9 × 10⁻⁴ 0.41 RB-1 + 3.6 3.49 3.2 × 10⁶ 2.3 × 10⁻⁴ 0.071 1.4 × 10⁶ 7 ×10⁻⁴ 0.48 YTE

The introduction of the YTE mutations in the Fc portion of the RB1 didnot alter the antibody's in vitro potency to neutralize RSV A and Bstrains. The in vitro neutralization potency for RSV A was 3 and 3.6ng/ml for RB1 and RB1+YTE respectively. The potencies for in vitroneutralization for RSV B were 1.7 and 3.49 ng/m for RB1 and RB1+YTErespectively. The kinetic constants as measured by Biocore were similarfor RB1 and RB1+YTE suggesting that introduction of YTE in the Fc regionof the antibody did not alter its antigen binding properties.

A non-GLP (Good Laboratory Practice) pharmacokinetics study wasconducted at New Iberia Research Center (UL Lafayette, La.). Eightbiologics-naïve male rhesus monkeys were randomized and assigned to oneof two study groups (n=4 per group). Each animal received a singleintravenous (i.v.) dose of RB1+YTE) or Motavizumab-YTE at 10 mg/kg.Blood samples were drawn prior to dosing on day 0, at 0.5, 1, 3, 8 and24 h after dosing, and at 1, 2, 3, 5, 7 and 10 days after dosing. AnECL-based immunoassays was used to quantify both RB1+YTE (Human×[RSV]mAb (RB1−YTE) IgG1/Kappa (CE)) and Motavizumab_YTE (Humanized×[RSV] mAbIgG1/Kappa) in rhesus monkey serum.

The assay used biotinylated mouse anti-human IgG kappa chain (BDBiosciences, San Jose, Calif.) as a capture reagent and sulfoTAG mouseanti-Human IgG Fc as a detection reagent (SouthernBiotech Birmingham,Ala.). The Lower Limit of Quantification (LLOQ) of the assay wasdetermined to be 1.37 ng/ml with Minimum Required Dilution (MRD) of 20.

Pharmacokinetics of RB1+YTE and Motavizumab-YTE were evaluated up to 10days in rhesus macaque dosed intravenously at 10 mg/kg using the sameimmunoassay to quantify RB1+YTE and Motavizumab-YTE. For each animal, anon-compartmental model was fitted for the serum concentration data ofeach animal using Phoenix Winnonlin 6.3 (Certara, N.J.) to estimate thearea under the curve (AUC0-10day). AUC0-10day was averaged across 4animals for each treatment group and reported as mean±standarddeviation. For RB1+YTE, AUC0-10day=1159±116 μg/mL*day and forMotavizumab-YTE, AUC0-10day=1381±63.0 μg/mL*day

RB1+YTE (depicted as RB1−YTE in the figure legend) and Motavizumab-YTEshowed similar serum concentration profiles and pharmacokinetics inrhesus macaque. See FIG. 7. RB1+YTE PK in NHP was also found to becomparable to motavizumab-YTE PK in cynomolgus monkey reported inliterature. See Dall'Acqua et al., 2006, J Biol Chem, 281:23514-24.

All references cited herein are incorporated by reference to the sameextent as if each individual publication, database entry (e.g. Genbanksequences or GeneID entries), patent application, or patent, wasspecifically and individually indicated to be incorporated by reference.This statement of incorporation by reference is intended by Applicants,pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and everyindividual publication, database entry (e.g. Genbank sequences or GeneIDentries), patent application, or patent, each of which is clearlyidentified in compliance with 37 C.F.R. § 1.57(b)(2), even if suchcitation is not immediately adjacent to a dedicated statement ofincorporation by reference. The inclusion of dedicated statements ofincorporation by reference, if any, within the specification does not inany way weaken this general statement of incorporation by reference.Citation of the references herein is not intended as an admission thatthe reference is pertinent prior art, nor does it constitute anyadmission as to the contents or date of these publications or documents.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

TABLE 7 Sequence Information SEQ ID NO: Description SEQUENCE 1RB1 H - CDR1 DSAMS 2 RB1 H - CDR2 FIKSKTYGGTKEYAASVKG 3 RB1 H - CDR3GAPYGGNSDYYYGLDV 4 RB1 L - CDR1 RTSQDVRGALA 5 RB1 L - CDR2 DASSLET 6RB1 L - CDR3 QQFLDFPFT 7 RB1 VHEVQLVESGGGLVRPGRSLRLSCTVSGFSFDDSAMSWVRQAPGKGLEWISFIKSKTYGGTKEYAASVKGRFTISRDDSKNIAYLQMNSLKTEDTAVYYCTRGAPYGGNSDYYYGLDVWGQGTTV T VSS 8 RB1 VL (patientDIQMTQSPSSLSASVGDRVTITCRTSQDVRGALAWYQQKPG isolated)KAPKLLIFDASSLETGVPSRFSGSGSGTVFTLTISSLQPED FAAYYCQQFLDFPFTFGQGTRLEIKRT 9RB1 VH (patient EVQLVESGGGLVRPGRSLRLSCTVSGFSFDDSAMSWVRQAP isolated)GKGLEWISFIKSKTYGGTKEYAASVKGRFTISRDDSKNIAYLQMNSLKTEDTAVYYCTRGAPYGGNSDYYYGLDVWGQGTTV IVSS 10 Leader sequenceMGWSCIILFLVATATGVHS 11 RB1 VH + leaderMGWSCIILFLVATATGVHSEVQLVESGGGLVRPGRSLRLSCTVSGFSFDDSAMSWVRQAPGKGLEWISFIKSKTYGGTKEYAASVKGRFTISRDDSKNIAYLQMNSLKTEDTAVYYCTRGAPY GGNSDYYYGLDVWGQGTTV T VSS 12RB1 VL + leader MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCRTSQDVRGALAWYQQKPGKAPKLLIFDASSLETGVPSRFSGSGSGTVFTLTISSLQPEDFAAYYCQQFLDFPFTFGQGTRL EIKRT 13 Heavy chainASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW constant domain-NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI IgG1CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 14 Kappa light chainVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV constant domainDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC 15Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACGGCC encoding RB1 VHAGGGCGGTCCCTGAGACTCTCCTGCACAGTTTCTGGATTCAGCTTTGACGACTCTGCTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATAAGTTTCATTAAAAGTAAAACTTATGGTGGGACAAAAGAATACGCCGCGTCTGTGAAAGGCAGGTTCACCATCTCAAGAGATGATTCCAAAAACATCGCCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTATTGTACTAGAGGGGCGCCTTACGGCGGTAACTCCGATTACTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTC ACTGTCTCCTCA 16 Nucleic acidGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATC encoding RB1 VLTGTAGGAGACAGAGTCACCATCACTTGCCGGACAAGTCAGGACGTTAGAGGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAACTCCTGATCTTTGATGCCTCCAGTTTGGAGACTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGTTTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAGCTTATTACTGTCAGCAGTTTCTTGATTTCCCTTTCACCTTCGGCCAGGGGACACGACTGGAAATCAAACGTACG 17 Nucleic acidGAGGTGCAGCTGGTCGAGAGCGGGGGGGGGCTGGTGCGGCC encoding RB1 VHTGGCAGGTCTCTGAGACTGAGCTGCACCGTGAGCGGCTTCT (codon optimized)CCTTTGACGATTCTGCCATGAGCTGGGTGCGGCAGGCTCCAGGCAAGGGACTGGAGTGGATCTCCTTCATCAAGTCTAAGACCTACGGCGGCACAAAGGAGTACGCCGCTTCCGTGAAGGGCCGGTTTACCATCAGCAGGGACGATTCCAAGAACATCGCCTATCTGCAGATGAACAGCCTGAAGACCGAGGACACAGCCGTGTACTATTGCACAAGAGGAGCTCCTTACGGAGGCAACAGCGACTACTATTACGGACTGGACGTGTGGGGACAGGGAACCACAGTG ACCGTGAGCTCC 18 Nucleic acidGACATTCAGATGACTCAGTCCCCTTCAAGTCTGAGCGCCTC encoding RB1 VLCGTGGGCGACAGAGTGACCATCACATGCCGGACCAGCCAGG (codon optimized)ATGTGCGGGGCGCCCTGGCTTGGTACCAGCAGAAGCCAGGCAAGGCCCCCAAGCTGCTGATCTTTGACGCTAGCTCCCTGGAGACCGGCGTGCCCTCCAGGTTTTCTGGCAGCGGCTCCGGCACAGTGTTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTTGCCGCTTACTATTGCCAGCAGTTCCTGGATTTCCCCTTCACCTTCGGCCAAGGCACACGGCTGGAGATCAAGAGGACC 19 Nucleic acidATGGGTTGGTCCTGTATTATCCTGTTCCTGGTCGCCACTGC encoding LeaderTACTGGGGTCCACTCA sequence heavy chain 20 Nucleic acidATGGGCTGGTCCTGTATTATCCTGTTCCTGGTGGCAACCGC encoding LeaderAACTGGTGTGCATAGC sequence light chain 21 Nucleic acidGCCTCTACAAAGGGCCCTAGCGTGTTCCCACTGGCTCCCTC encoding HeavyTTCCAAGTCTACCAGCGGAGGAACAGCCGCTCTGGGATGTC chain constantTGGTGAAGGATTACTTCCCAGAGCCCGTGACCGTGTCCTGG domain-AACTCTGGCGCCCTGACCAGCGGAGTGCACACATTTCCAGC IgG1TGTGCTGCAGTCCTCTGGCCTGTATTCCCTGAGCTCCGTGGTGACCGTGCCCTCTAGCTCCCTGGGCACCCAGACATACATCTGTAACGTGAATCACAAGCCAAGCAATACAAAGGTGGACAAGAAGGTCGAGCCCAAGTCCTGTGATAAGACCCACACATGCCCCCCTTGTCCTGCTCCAGAGCTGCTGGGAGGACCTAGCGTGTTCCTGTTTCCACCCAAGCCTAAGGACACCCTGATGATCTCTAGGACCCCCGAGGTGACATGCGTGGTGGTGGACGTGAGCCACGAGGATCCTGAGGTGAAGTTTAACTGGTACGTCGATGGCGTGGAGGTGCACAATGCCAAGACAAAGCCCAGAGAGGAGCAGTATAACTCCACCTACCGGGTGGTGTCTGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAATAAGGCCCTGCCCGCTCCTATCGAGAAGACCATCTCTAAGGCCAAGGGCCAGCCTAGGGAGCCACAGGTGTATACACTGCCTCCATCCAGAGACGAGCTGACCAAGAACCAGGTGTCTCTGACATGTCTGGTGAAGGGCTTCTACCCTTCTGATATCGCCGTGGAGTGGGAGAGCAATGGCCAGCCAGAGAACAATTATAAGACCACACCCCCTGTGCTGGACAGCGATGGCTCCTTCTTTCTGTACAGCAAGCTGACCGTGGATAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGTTCTGTGATGCACGAAGCCCTGCACAATCACTACACTCAGAAGAGCCTGTCCCTGTCACCT GGTAA 22 Nucleic acidGTGGCCGCTCCCTCCGTGTTTATCTTCCCCCCTTCTGACGA encoding KappaGCAGCTGAAGTCTGGCACAGCTAGCGTGGTGTGCCTGCTGA light chainACAATTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTG constant domainGATAACGCTCTGCAGTCTGGCAATAGCCAGGAGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCCTCTACCCTGACACTGTCTAAGGCCGATTACGAGAAGCACAAGGTGTATGCTTGTGAAGTCACCCACCAGGGGCTGAGTTCACCAGT CACCAAGTCATTCAATCGGGGCGAGTGC23 RB1 + YTE Heavy EVQLVESGGGLVRPGRSLRLSCTVSGFSFDDSAMSWVRQAP ChainGKGLEWISFIKSKTYGGTKEYAASVKGRFTISRDDSKNIAYLQMNSLKTEDTAVYYCTRGAPYGGNSDYYYGLDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 24 Nucleic acidGAGGTGCAGCTGGTGGAATCCGGCGGCGGACTGGTCAGACC encoding RB1 +TGGCAGATCCCTGAGGCTCAGCTGTACCGTGAGCGGCTTCA YTEGCTTCGACGACTCCGCCATGAGCTGGGTGAGACAGGCCCCT Heavy ChainGGCAAGGGCCTGGAGTGGATCAGCTTCATCAAGAGCAAAAC (codon optimized)CTATGGCGGAACCAAGGAATACGCCGCCTCCGTGAAGGGCAGGTTCACCATTTCCAGGGACGACAGCAAGAACATCGCTTACCTCCAGATGAACTCCCTCAAGACCGAGGATACCGCCGTGTATTATTGCACCAGAGGCGCCCCCTACGGCGGCAATTCCGACTATTACTACGGCCTGGATGTCTGGGGCCAAGGCACAACAGTGACCGTGAGCTCCGCTAGCACCAAGGGACCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACAAGCGGAGGAACAGCCGCCCTCGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCTGTGACAGTCAGCTGGAATAGCGGCGCTCTGACCAGCGGCGTCCACACCTTTCCCGCTGTCCTGCAGAGCTCCGGCCTGTACAGCCTGTCCTCCGTGGTCACAGTGCCCTCCTCCAGCCTGGGCACACAAACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTCGAACCCAAATCCTGTGACAAGACCCACACATGCCCCCCCTGCCCCGCCCCTGAGCTGCTGGGCGGCCCTTCCGTGTTCCTGTTCCCTCCCAAGCCCAAGGATACCCTGTATATCACCAGAGAACCCGAGGTGACCTGTGTGGTGGTCGACGTCAGCCACGAAGATCCTGAGGTCAAGTTCAACTGGTATGTGGACGGCGTGGAGGTGCATAACGCCAAAACCAAGCCCAGGGAGGAACAGTATAACAGCACCTACAGGGTGGTGTCCGTCCTGACCGTGCTGCACCAGGACTGGCTGAACGGAAAGGAGTACAAATGTAAGGTCAGCAACAAAGCCCTGCCCGCTCCTATCGAAAAGACCATCTCCAAGGCCAAAGGCCAGCCCAGAGAACCCCAGGTGTACACCCTGCCCCCTAGCAGAGACGAGCTGACCAAAAACCAGGTCTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAAAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTCGACAGCGATGGCAGCTTCTTTCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAACAAGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCTCTGCACAACCACTATACCCAGAAGTCCCTG AGCCTCAGCCCCGGAAAATGA 25RB1 + YTE Light DIQMTQSPSSLSASVGDRVTITCRTSQDVRGALAWYQQKPG ChainKAPKLLIFDASSLETGVPSRFSGSGSGTVFTLTISSLQPEDFAAYYCQQFLDFPFTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 26 Nucleic acidGACATTCAGATGACTCAGTCCCCTTCAAGTCTGAGCGCCTC encoding RB1 +CGTGGGCGACAGAGTGACCATCACATGCCGGACCAGCCAGG YTEATGTGCGGGGCGCCCTGGCTTGGTACCAGCAGAAGCCAGGC Light ChainAAGGCCCCCAAGCTGCTGATCTTTGACGCTAGCTCCCTGGAGACCGGCGTGCCCTCCAGGTTTTCTGGCAGCGGCTCCGGCACAGTGTTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTTGCCGCTTACTATTGCCAGCAGTTCCTGGATTTCCCCTTCACCTTCGGCCAAGGCACACGGCTGGAGATCAAGAGGACCGTGGCCGCTCCCTCCGTGTTTATCTTCCCCCCTTCTGACGAGCAGCTGAAGTCTGGCACAGCTAGCGTGGTGTGCCTGCTGAACAATTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCTCTGCAGTCTGGCAATAGCCAGGAGTCCGTGACCGAGCAGGACTCTAAGGATAGCACATATTCCCTGTCCTCTACCCTGACACTGTCTAAGGCCGATTACGAGAAGCACAAGGTGTATGCTTGTGAAGTCACCCACCAGGGGCTGAGTTCACCAGTC ACCAAGTCATTCAATCGGGGCGAGTGC

1.-25. (canceled)
 26. An isolated antibody that binds or interacts withan epitope on human RSV pre-fusion or human RSV post-fusion F proteins,wherein the epitope on the human RSV F protein comprises the followingamino acids set forth in Genbank Accession Number AAR14266: anasparagine at position 426 (Asn 426), an arginine at position 429 (Arg429), an isoleucine at position 432 (Iso 432), a lysine at position 433(Lys 433), and a lysine at position 445 (Lys 445), wherein the isolatedantibody comprises a light chain variable region and a heavy chainvariable region, and wherein the isolated antibody does not comprise: aheavy chain variable region having the amino acid sequence set forth inSEQ ID NO:
 9. 27. The isolated antibody of claim 26, comprising: a heavychain comprising the heavy chain variable region of SEQ ID NO: 7 or avariant thereof comprising up to 30 amino acid substitutions, and/or alight chain comprising the light chain variable region of SEQ ID NO: 8comprising up to 12 amino acid substitutions.
 28. The isolated antibodyof claim 27, wherein the isolated antibody comprises a constant domainmodified to increase the isolated antibody's biological half-life. 29.The isolated antibody of claim 28, wherein the constant domain modifiedto increase the isolated antibody's biological half-life corresponds toa) one or more of the mutations T252L, T254S, T256F, b) a mutationwithin the CH1 or CL region to contain a salvage receptor bindingepitope taken from two loops of a CH2 domain of an Fc region of an IgG,or c) the triple mutation M252Y/S254T/T256E (YTE).
 30. The isolatedantibody of claim 29, wherein the constant domain modified to increasethe isolated antibody's biological half-life corresponds to the triplemutation M252Y/S254T/T256E (YTE).
 31. The isolated antibody of claim 26,wherein the isolated antibody binds to human RSV F protein through allof the following interactions: 1) the light chain CDR3 loop, throughresidues Phe 91 and Leu 92, interacts with the side chain of Arg 429 ofhuman RSV F protein through the formation of two hydrogen bonds betweenthe carbonyl oxygens of Phe 91 and Leu 92 in the CDR3 loop and theguanidino nitrogens of Arg 429 of human RSV F protein; 2) the lightchain CDR2 loop, through residues Asp 50 and Glu 55, forms hydrogenbonds with Asn 426 and Lys 445 of human RSV F protein; 3) the heavychain CDR3 loop, through residues Tyr 104 and Tyr 110, form a surfacefor van der Waals interaction with Ile 432 on human RSV F protein; 4)the heavy chain CDR3 loop, through Asn 107, forms a hydrogen bond withLys 433 of human RSV F protein; and 5) the light chain packs against Glu161 and Ser 182 of the neighboring monomer of a RSV pre-fusion trimer.32. A composition comprising the isolated antibody of claim 26 and apharmaceutically acceptable carrier or diluent.
 33. A method ofpreventing or treating an RSV infection in a human subject, comprisingadministering to the subject an effective amount of the isolatedantibody of claim
 26. 34. An isolated antibody that binds or interactswith an epitope on human RSV pre-fusion or human RSV post-fusion Fproteins, wherein the epitope on the human RSV F protein comprises thefollowing amino acids set forth in Genbank Accession Number AAR14266, anasparagine at position 426 (Asn 426), an arginine at position 429 (Arg429), an isoleucine at position 432 (Iso 432), a lysine at position 433(Lys 433), and a lysine at position 445 (Lys 445), wherein the isolatedantibody comprises a light chain variable region and a heavy chainvariable region, and comprises at least 90%, 95%, 96%, 97%, 98% or 99%sequence identity with the heavy chain variable region of SEQ ID NO: 7or the light chain variable region of SEQ ID NO: 8, and wherein theisolated antibody does not comprise a heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO:
 9. 35. The isolatedantibody of claim 34, wherein the isolated antibody comprises a constantdomain modified to increase the antibody's biological half-life.
 36. Theisolated antibody of claim 35, wherein the constant domain modified toincrease the isolated antibody's biological half-life corresponds to a)one or more of the mutations T252L, T254S, T256F, b) a mutation withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, or c) thetriple mutation M252Y/S254T/T256E (YTE).
 37. The isolated antibody ofclaim 36, wherein the constant domain modified to increase the isolatedantibody's biological half-life corresponds to the triple mutationM252Y/S254T/T256E (YTE).
 38. The isolated antibody of claim 34, whereinthe isolated antibody has at least one of the following characteristics:(i) binds to a human RSV pre-fusion F protein with a Kd value of about1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmon resonance;(ii) binds to a human RSV post-fusion F protein with a Kd value of about1×10⁻⁹ M to about 1×10⁻¹¹ M as determined by surface plasmon resonance;or (iii) binds to a human RSV pre-fusion F protein with a Kd value ofabout 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmonresonance and binds to a human RSV post-fusion F protein with a Kd valueof about 1×10⁻⁹ M to about 1×10⁻¹¹ M as determined by surface plasmonresonance.
 39. The isolated antibody of claim 34, wherein the isolatedantibody binds to human RSV F protein through all of the followinginteractions: 1) the light chain CDR3 loop, through residues Phe 91 andLeu 92, interacts with the side chain of Arg 429 of human RSV F proteinthrough the formation of two hydrogen bonds between the carbonyl oxygensof Phe 91 and Leu 92 in the CDR3 loop and the guanidino nitrogens of Arg429 of human RSV F protein; 2) the light chain CDR2 loop, throughresidues Asp 50 and Glu 55, forms hydrogen bonds with Asn 426 and Lys445 of human RSV F protein; 3) the heavy chain CDR3 loop, throughresidues Tyr 104 and Tyr 110, form a surface for van der Waalsinteraction with Ile 432 on human RSV F protein; 4) the heavy chain CDR3loop, through Asn 107, forms a hydrogen bond with Lys 433 of human RSV Fprotein; and 5) the light chain packs against Glu 161 and Ser 182 of theneighboring monomer of a RSV pre-fusion trimer
 40. A compositioncomprising the isolated antibody of claim 34 and a pharmaceuticallyacceptable carrier or diluent.
 41. A method of preventing or treating anRSV infection in a human subject, comprising administering to thesubject an effective amount of the isolated antibody of claim
 34. 42. Anisolated antibody that cross-competes with an antibody or antigenbinding fragment comprising a light chain variable region having theamino acid sequence set forth in SEQ ID NO: 8 and a heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO: 7 forbinding to an epitope of human RSV pre-fusion or post-fusion F proteinhaving the amino acid sequence set forth in Genbank Accession NumberAAR14266, wherein the isolated antibody comprises a light chain variableregion and a heavy chain variable region, and wherein the isolatedantibody has at least one of the following characteristics: wherein theisolated antibody comprises at least 90%, 95%, 96%, 97%, 98% or 99%sequence identity with the heavy chain variable region of SEQ ID NO: 7or the light chain variable region of SEQ ID NO: 8, and wherein theisolated antibody does not comprise a heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO:
 9. 43. The isolatedantibody of claim 42, wherein the isolated antibody comprises a constantdomain modified to increase the antibody's biological half-life.
 44. Theisolated antibody of claim 43, wherein the constant domain modified toincrease the antibody's biological half-life corresponds to a) one ormore of the mutations T252L, T254S, T256F, b) a mutation within the CH1or CL region to contain a salvage receptor binding epitope taken fromtwo loops of a CH2 domain of an Fc region of an IgG, or c) the triplemutation M252Y/S254T/T256E (YTE).
 45. The isolated antibody of claim 44,wherein the constant domain modified to increase the antibody'sbiological half-life corresponds to the triple mutationM252Y/S254T/T256E (YTE).
 46. The isolated antibody of claim 42, whereinthe isolated antibody binds to human RSV F protein through all of thefollowing interactions: 1) the light chain CDR3 loop, through residuesPhe 91 and Leu 92, interacts with the side chain of Arg 429 of human RSVF protein through the formation of two hydrogen bonds between thecarbonyl oxygens of Phe 91 and Leu 92 in the CDR3 loop and the guanidinonitrogens of Arg 429 of human RSV F protein; 2) the light chain CDR2loop, through residues Asp 50 and Glu 55, forms hydrogen bonds with Asn426 and Lys 445 of human RSV F protein; 3) the heavy chain CDR3 loop,through residues Tyr 104 and Tyr 110, form a surface for van der Waalsinteraction with Ile 432 on human RSV F protein; 4) the heavy chain CDR3loop, through Asn 107, forms a hydrogen bond with Lys 433 of human RSV Fprotein; and 5) the light chain packs against Glu 161 and Ser 182 of theneighboring monomer of a RSV pre-fusion trimer.
 47. The isolatedantibody of claim 42, wherein the isolated antibody has at least one ofthe following characteristics: (i ) binds to a human RSV pre-fusion Fprotein with a Kd value of about 1×10⁻⁹ M to about 1×10⁻¹² M asdetermined by surface plasmon resonance; (ii) binds to a human RSVpost-fusion F protein with a Kd value of about 1×10⁻⁹ M to about 1×10⁻¹¹M as determined by surface plasmon resonance; or (iii) binds to a humanRSV pre-fusion F protein with a Kd value of about 1×10⁻⁹ M to about1×10⁻¹² M as determined by surface plasmon resonance and binds to ahuman RSV post-fusion F protein with a Kd value of about 1×10⁻⁹ M toabout 1×10⁻¹¹ M as determined by surface plasmon resonance.
 48. Anisolated antibody comprising a heavy chain variable region comprising atleast 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7, a lightchain variable region comprising at least 90%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO: 8, and a constant domain modified to increase theantibody's biological half-life.
 49. The isolated antibody of claim 48,wherein the constant domain corresponds to a) one or more of themutations T252L, T254S, T256F, b) a mutation within the CH1 or CL regionto contain a salvage receptor binding epitope taken from two loops of aCH2 domain of an Fc region of an IgG, or c) the triple mutationM252Y/S254T/T256E (YTE).
 50. The isolated antibody of claim 49, whereinthe constant domain of the isolated antibody corresponds to the triplemutation M252Y/S254T/T256E (YTE).
 51. A composition comprising theisolated antibody of claim 48 and a pharmaceutically acceptable carrieror diluent.
 52. A method of preventing or treating an RSV infection in ahuman subject, comprising administering to the subject an effectiveamount of the isolated antibody of claim 48.